U.S. patent application number 14/897885 was filed with the patent office on 2016-06-09 for polydendrons.
This patent application is currently assigned to The Univeristy of Liverpool. The applicant listed for this patent is The Univeristy of Liverpool. Invention is credited to Samuel Auty, Pierre Chambon, Andrew Dwyer, Marco Giardiello, Fiona Hatton, Andrew Owen, Steven Rannard, Hannah Rogers, Lee Tatham.
Application Number | 20160159940 14/897885 |
Document ID | / |
Family ID | 48914593 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160159940 |
Kind Code |
A1 |
Rannard; Steven ; et
al. |
June 9, 2016 |
POLYDENDRONS
Abstract
A method of preparing a non-gelled branched vinyl polymer
scaffold carrying dendrons, comprising the living or controlled
polymerization of a monofunctional vinyl monomer and a difunctional
vinyl monomer, using a dendron initiator and at least one further
initiator.
Inventors: |
Rannard; Steven; (Liverpool,
GB) ; Owen; Andrew; (Liverpool, GB) ; Rogers;
Hannah; (Liverpool, GB) ; Giardiello; Marco;
(Liverpool, GB) ; Hatton; Fiona; (Liverpool,
GB) ; Chambon; Pierre; (Liverpool, GB) ; Auty;
Samuel; (Liverpool, GB) ; Dwyer; Andrew;
(Liverpool, GB) ; Tatham; Lee; (Liverpool,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Univeristy of Liverpool |
|
|
|
|
|
Assignee: |
The Univeristy of Liverpool
Liverpool
GB
The Univeristy of Liverpool
Liverpool
GB
|
Family ID: |
48914593 |
Appl. No.: |
14/897885 |
Filed: |
June 13, 2014 |
PCT Filed: |
June 13, 2014 |
PCT NO: |
PCT/GB2014/051822 |
371 Date: |
December 11, 2015 |
Current U.S.
Class: |
514/772.4 ;
526/209; 526/304 |
Current CPC
Class: |
A61K 9/5138 20130101;
C08G 83/002 20130101; A61K 49/0041 20130101; A61K 49/0054 20130101;
C08F 2438/01 20130101; A61K 49/0028 20130101; C08F 220/58 20130101;
C08F 220/28 20130101; C08L 2203/02 20130101; C08F 222/1006
20130101; C08F 2/38 20130101 |
International
Class: |
C08F 2/38 20060101
C08F002/38; C08F 220/58 20060101 C08F220/58; A61K 9/51 20060101
A61K009/51 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 14, 2013 |
GB |
1310655.4 |
Claims
1. A method of preparing a non-gelled branched vinyl polymer
scaffold carrying dendrons, comprising the living or controlled
polymerization of a monofunctional vinyl monomer and a difunctional
vinyl monomer, using a dendron initiator and at least one further
initiator.
2. The method as claimed in claim 1 wherein the living
polymerization is ATRP.
3. The method as claimed in claim 1 wherein the molar ratio of
difunctional vinyl monomer to initiators is less than 1.
4. The method as claimed in claim 1 wherein the further initiator
is selected from or comprises one or more of the following: a small
molecule, a drug, an active pharmaceutical ingredient, a polymer, a
peptide, a sugar, a dendron, a moiety which carries or can carry a
drug, an anionic functional group, a cationic functional group, a
moiety which enhances solubility, a moiety which prolongs residence
time within the body, a moiety which enhances stability of a drug
or other active material, a moiety which reduces macrophage uptake,
a moiety which enhances controlled release, a moiety which enhances
drug transport, or a moiety which enhances drug targeting.
5. The method as claimed in claim 1 wherein the further initiator
comprises a PEG group.
6. The method as claimed in claim 1 wherein the dendron initiator
comprises a generation 1 dendron.
7. The method as claimed in claim 6 wherein the first generation
branches are identical.
8. The method as claimed in claim 1 wherein the dendron initiator
comprises a generation 2 dendron.
9. The method as claimed in claim 8 wherein the second generation
branches are identical.
10. The method as claimed in claim 1 wherein one or more of the
initiators comprises a functional group allowing
post-functionalization.
11. The method as claimed in claim 1 followed by nanoprecipitation
to form nanoparticles.
12. The method as claimed in claim 1 wherein the monofunctional
vinyl monomer and/or the difunctional vinyl monomer comprise a
methacrylate.
13. A product obtained by the method of claim 1.
14. A non-gelled branched vinyl polymer scaffold carrying one type
of dendron moiety and a further moiety.
15. The scaffold as claimed in claim 14 which is an atom transfer
radical polymerized material.
16. The scaffold as claimed in claim 14 wherein the further moiety
is selected from one or more of the following: a small molecule, a
drug, an active pharmaceutical ingredient, a polymer, a peptide, a
sugar, a dendron, a moiety which carries or can carry a drug, an
anionic functional group, a cationic functional group, a moiety
which enhances solubility, a moiety which prolongs residence time
within the body, a moiety which enhances stability of a drug or
other active material, a moiety which reduces macrophage uptake, a
moiety which enhances controlled release, a moiety which enhances
drug transport, or a moiety which enhances drug targeting.
17. The scaffold as claimed in claim 14 wherein the further moiety
comprises a PEG group.
18. The scaffold as claimed in claim 14 wherein the dendron
initiator comprises a generation 1 dendron.
19. The scaffold as claimed in claim 18 wherein the first
generation branches are identical.
20. The scaffold as claimed in claim 14 wherein the dendron
initiator comprises a generation 2 dendron.
21. The scaffold as claimed in claim 20 wherein the second
generation branches are identical.
22. The scaffold as claimed in claim 14 wherein one or more of the
initiators comprises a functional group allowing
post-functionalization.
23. A nanoparticle comprising the scaffold as claimed in claim
14.
24. A pharmaceutical composition comprising the product as claimed
in claim 13 and a pharmaceutically acceptable diluent.
25. The pharmaceutical composition as claimed in claim 24 which is
formulated for oral, parenteral, topical or ocular
administration.
26-30. (canceled)
31. A method of treatment comprising administration of the product
as claimed in claim 13 to a patient in need thereof.
Description
[0001] The present invention relates to nanomaterials, in
particular nanomaterials having hybrid structures comprising a
branched vinyl polymer scaffold together with dendritic components.
The present invention is particularly, though not exclusively,
concerned with such hybrid materials from the perspective of
medical applications, for example the carrying and delivering of
drugs and other medically useful materials, the enhancement of
therapeutic and diagnostic properties, and improved or more
efficient or cost-effective formulations.
[0002] Dendrimers have been extensively studied in this context,
amongst many other contexts. The word "dendrimer" was coined in the
early 1980s, following work on cascade chemistry and arborols, to
describe polymers which contain dendrons. A "dendron" is a
tree-like, repeatedly-branched, moiety. Thus, a dendron is a
wedge-shaped dendritic fragment of a dendrimer. Typically,
dendrimers have ordered, symmetrical architectures. A dendrimer
comprises a core from which several dendrons branch outwards, to
form a three-dimensional, usually spherical structure.
[0003] Dendrimers can be prepared by step-wise divergent or
convergent growth. Divergent procedures start at the core of the
dendrimer and grow outwards. Convergent procedures prepare dendrons
first and then couple the dendrons together. In convergent
procedures, the dendrons are typically coupled together at their
focal points (i.e. at the base of the "tree", or the apex of the
dendritic wedge) via chemically addressable groups.
[0004] For a nanomaterial to carry and deliver a drug or other
biologically useful material, it is necessary for it to exhibit
suitable properties in aqueous media and to have suitable domains
to encapsulate the drug (which, for most drugs, need to be
hydrophobic domains) and/or means of conjugating, bonding or
otherwise associating with the drug. It is also advantageous for
the nanomaterial to be able to carry a high "payload" of drug.
Dendrimers satisfy these requirements. Due to their repeatedly
branched iterative nature, they are large compared to non-polymeric
active molecules and contain a large number of surface groups, and
can therefore encapsulate, and/or be conjugated to, a large amount
of material. Whilst they can be made from all kinds of chemical
building blocks, they commonly comprise organic chains which
provide hydrophobic microenvironments for drugs or other organic
molecules. At the same time they can be stable in aqueous media so
that drugs or other hydrophobic materials can be delivered within
the body.
[0005] Whilst dendrimers have many interesting properties and
promising features, they also have significant disadvantages.
Dendrimer syntheses are lengthy and costly. The production of
ideally branched structures requires multiple repeated steps of
synthesis, purification and characterisation. Maintaining a 100%
degree of branching generates complexity and takes time and
requires very controlled reaction conditions. Even with high levels
of successful recovery between steps, the compound effect after
several steps means that the overall mass recovery suffers
significantly. Whilst convergent methods are better than divergent
methods from the viewpoint of ease and speed of procedure, they are
still arduous, and other problems beset convergent methods, for
example steric difficulties hindering coupling.
[0006] Geometric realities of iterative branching mean that the
crowding constraints at the surface of the dendrimer sphere limit
the size of the nanomaterials. Therefore dendrimers typically have
a maximum size of about 10 nm. This limits the amount of material
they can carry.
[0007] Further description of dendrimers and their structures,
preparation and applications, can be found in numerous articles
including: S. M. Grayson and J. M. Frechet, Chem. Rev. 2001, 101,
3819-3867; H. Frauenrath, Prog. Polym. Sci 2005, 325-384; F.
Aulenta, W. Hayes and S. Rannard, European Polymer Journal 2003,
39, 1741-1771; E. R. Gillies and J. M. J. Frechet, Drug Discovery
Today, 2005, 10, 1, 35-43; and S. H. Medina and M. E. H. El-Sayed,
Chem. Rev. 2009, 109, 3141-3157.
[0008] From a first aspect the present invention provides a method
of preparing a non-gelled branched vinyl polymer scaffold carrying
dendrons, comprising the living or controlled polymerization of a
monofunctional vinyl monomer and a difunctional vinyl monomer,
using more than one initiator, at least one of which is a dendron
initiator.
[0009] From a second aspect the present invention provides a
non-gelled branched vinyl polymer scaffold carrying more than one
type of moiety, at least one of which is a dendron moiety.
[0010] Thus the present invention provides products which can be
referred to as "polydendrons" because they contain a plurality of
dendrons. The dendrons may be the same or different. Polydendrons
retain the advantages of dendrimers without having their
disadvantages of cost, complexity and arduous synthesis. Instead of
the dendritic structure extending all the way to the centre, the
core is a tuneable and cost-effective non-gelled branched vinyl
polymer scaffold. The polydendrons typically take the form of units
(which optionally are approximately spherical) with a large number
of external surface dendron groups and with the vinyl scaffolds
typically being present predominantly in the centre of the
units.
[0011] The non-gelled branched vinyl polymer scaffolds of the
present invention exhibit good solubility and low viscosity. They
can be contrasted with polymer structures which are insoluble
and/or exhibit high viscosity, such as extensively crosslinked
insoluble polymer networks, high molecular weight linear polymers,
or microgels.
[0012] The products can be made by, but are not limited to being
made by, living polymerization, controlled polymerization or
chain-growth polymerization. Several types of living and controlled
polymerization are known in the art and suitable for use in the
present invention. A preferred type of living polymerization is
Atom Transfer Radical Polymerization (ATRP), however other
techniques such as Reversible Addition-Fragmentation chain-Transfer
(RAFT) and Nitroxide Mediated Polymerisation (NMP) or conventional
free-radical polymerization controlled by the deliberate addition
of chain-transfer agents are also suitable syntheses.
[0013] The skilled person is aware of techniques to provide
branched but non-gelled vinyl polymer scaffolds. For example,
suitable procedures are described in WO 2009/122220; N. O'Brien, A.
McKee, D. C. Sherrington, A. T. Slark and A. Titterton, Polymer
2000, 41, 6027-6031; T. He, D. J. Adams, M. F. Butler, C. T. Yeoh,
A. I. Cooper and S. P. Rannard, Angew. Chem. Int. Ed. 2007, 46,
9243-9247; V. Butun, I. Bannister, N. C. Billingham, D. C.
Sherrington and S. P. Armes, Macromolecules 2005, 38, 4977-4982; I.
Bannister, N. C. Billingham, S. P. Armes, S. P. Rannard and P.
Findlay, Macromolecules 2006, 39, 7483-7492; and R. A. Slater, T. O
McDonald, D. J. Adams, E. R. Draper, J. V. M. Weaver and S. P.
Rannard, Soft Matter 2012, 8, 9816-9827. The non-gelled and soluble
products of the present invention are different to materials
disclosed in L. A. Connal, R. Vestberg, C J. Hawker and G. G. Qiao,
Macromolecules 2007, 40, 7855-7863 which comprise multiple
cross-linking in a gelled network.
[0014] The polymerization of each vinyl polymer chain starts at an
initiator. Polymerization of monofunctional vinyl monomers leads to
linear polymer chains. Copolymerization with difunctional vinyl
monomers leads to branching between the chains. In order to control
branching and prevent gelation there should be less than one
effective brancher (difunctional vinyl monomer) per chain. Under
certain conditions, this can be achieved by using a molar ratio of
brancher to initiator of less than one: this assumes that the
monomer (i.e. the monofunctional vinyl monomer) and the brancher
(i.e. the difunctional vinyl monomer) have the same reactivity,
that there is no intramolecular reaction, that the two
functionalities of the brancher have the same reactivity, and that
reactivity remains the same even after part-reaction. Of course,
the systems and conditions may be different, but the skilled person
understands how to control the reaction and determine without undue
experimentation how a non-gelled structure may be achieved. For
example, under dilute conditions some branchers form intramolecular
cycles which limit the number of branchers that branch between
chains even if the molar ratio of brancher to initiator (i.e.
polymer chain) is higher than 1:1 in the reaction.
[0015] In the present invention, dendrons are used as
macromolecular initiators. In order to be able to initiate
polymerization, the dendrons must bear suitable reactive
functionality. For example, in ATRP, convenient and effective
initiators include alkyl halides (e.g. alkyl bromides), and so
dendrons which carry halides at their focal points can act as
initiators. In this scenario, propagation starts at the apex of the
dendron "wedge". The skilled person is well aware of the types of
components and reagents which are used in ATRP and other living or
controlled polymerizations, and hence the type of functionality
which must be present on or introduced to dendrons for them to act
as initiators.
[0016] One possible way of introducing bromo groups to dendrons is
to functionalize dendron alcohols with alpha-bromoisobutyryl
bromide. There are however many other ways of functionalizing
dendrons so that they can act as initiators and other types of
functionality which will initiate polymerization. The concept of a
dendron initiator is applicable to all suitable types of
polymerization and the functionality can be varied as
necessary.
[0017] There is no particular limitation regarding the type of
dendron that can be used, or the chemistry used to prepare the
dendrons. In some scenarios it is desirable to have particular
groups present at the surface (i.e. at the tips of the "branches"
of the dendron), and these may be incorporated during the synthesis
of the dendron. The dendrons are preferably non-vinyl.
[0018] Any suitable coupling chemistry may be used to build up the
dendrons. In one example, amines and alcohols may be coupled
together, for example using carbonyldiimidazole. This is, however,
merely one example and numerous other coupling methods are
possible.
[0019] If exclusively one type of dendron initiator were used then
in the resultant hybrid branched product one end of each vinyl
polymer chain would bear that dendron.
[0020] In contrast, an essential feature of the present invention
is that mixed initiators are used, in other words not only a
dendron initiator but also at least one further initiator (which
may be a different type of dendron initiator, or alternatively an
initiator other than a dendron initiator) is used. This allows
considerable advantages in terms of varying the composition and the
properties of the resultant polydendron structure.
[0021] The present invention resides in the combination of features
which work well together. The branched vinyl polymer methodology is
intermingled with the use of mixed initiators including at least
one dendron initiator. The way in which the living or controlled
polymerization occurs means that the different initiators are
distributed statistically and evenly around the surface of the
non-gelled branched vinyl polymer scaffold. Some polymer chains
will have one type of initiator at one end whereas other polymer
chains will have another type at their end. There may be two types
of initiator, or more, e.g. three or four or more, and therefore
the multiplicity of types of end group may be two or more.
[0022] The vinyl polymer core is easily tuneable and very
cost-effective. Different types of monomers, with different
properties (e.g. differing solubility properties) may be used. The
methodology allows a sizeable scaffold to be built, and the
molecular weight and size can be controlled by choice of particular
monomers (a wide range can be used) and reaction conditions, for
example the ratio of initiator to monomer.
[0023] The material is non-gelled and therefore soluble. At the
same time the use of mixed initiators allows further tuneability
and flexibility. There are synergistic advantages: for example the
use of dendrons and other moieties as initiators means that they do
not need to be introduced separately but instead are used as
reagents within an already very efficient and convenient
polymerization process. The process conveniently and
cost-effectively results in the different types of initiators being
distributed throughout the materials. The initiators themselves are
relatively easy to synthesize. Regarding the need for the
initiators to have suitable means and functionality to initiate
polymerization, the considerations described above in relation to
the dendron initiators apply mutatis mutandis to the at least one
further initiator(s).
[0024] The living or controlled polymerization methodology
inherently allows control in the synthesis of the polymeric
scaffold. For example, ATRP and other techniques are robust and
flexible in being suitable for use with a large variety of
functional groups and in avoiding unwanted side reactions. The size
and dispersity of the products can be controlled. The monomer units
are usually homogeneously distributed between the initiator
molecules and therefore the chain length, and hence the molecular
weight, can be controlled. The conditions can be controlled to
result in materials having low polydispersity indexes when forming
linear polymers, i.e. mixtures wherein the individual components
have approximately the same size. This is particularly useful in
the present invention as the individual chains comprising the
branched structure (i.e. the primary chains) have similar chain
lengths. The resulting branched polymers of the invention have a
distribution of structures with varying numbers of linear chains
connected to form the branched architectures.
[0025] The use of at least one further initiator, in addition to
the dendron initiator, within the living or controlled
polymerization methodology, brings further advantages. The further
initiator alters the properties of the polydendron, for example the
solubility, hydrophilicity, hydrophobicity, aggregation, size,
reactivity, stability, degradability, therapeutic, diagnostic,
biological transport, plasma residence time, cell interaction, drug
compatibility, stimulus response, targeting and/or imaging
characteristics.
[0026] The further initiator may comprise or be derived from one or
more of the following: a small molecule, a drug, an active
pharmaceutical ingredient, a polymer, a peptide, a sugar, a
dendron, a moiety which carries or can carry a drug, an anionic
functional group, a cationic functional group, a moiety which
enhances solubility (for example, of the polydendron within aqueous
systems, or of a drug or other carried material), a moiety which
prolongs residence time within the body, a moiety which enhances
stability of a drug or other active material, a moiety which
reduces macrophage uptake, a moiety which enhances controlled
release, a moiety which enhances drug transport, or a moiety which
enhances drug targeting.
[0027] The initiator may be a macroinitiator, for example a
macroinitiator prepared by synthesis from one or more monomer (e.g.
a water soluble monofunctional monomer), or a macroinitiator
prepared by modification of a pre-synthesized polymer. The
macroinitiator may be a copolymer, i.e. may comprise a polymer made
from at least two monomers, e.g. monofunctional monomers. The
macroinitiator may further be selected from natural polymers, for
example water soluble or partially soluble polymers, e.g.
polysaccharides, polypeptides or proteins.
[0028] Each type of initiator may fall within one or more than one
of the above definitions; for example the initiator may be a
dendron and may also carry a drug. The initiator may also be a
pro-drug, releasing a moiety that becomes pharmacologically active
after a further process within the body.
[0029] The present inventors have been surprised at how effective
the use of mixed initiators is, in allowing a range of properties
to be controlled and tuned. As described in more detail below, they
have observed: that the surface chemistry can be varied widely
across a hydrophobic--amphiphilic--hydrophilic spectrum; that the
encapsulation environment can be varied significantly; that the
salt stability can be controlled; and that transcellular
permeability (in an in vitro model) can be tuned and improved.
[0030] In view of the drug delivery capabilities, from further
aspects the present invention also provides pharmaceutical
compositions comprising the products of the present invention, and
allows enhancements in terms of medical administration
possibilities.
[0031] For example, the surprisingly effective way in which the
polydendrons interact controllably with, and transport encapsulated
materials through, model gut-epithelium, is relevant to oral
delivery applications. Materials of this type are also useful
within parenteral administration such as intravenous, subcutaneous
and intramuscular injection.
[0032] Polyethylene glycol (PEG) groups are advantageous for use in
the initiators of the present invention. In comparison to
polydendrons which carry dendrons alone, polydendrons which carry
not only dendrons but also PEG groups exhibit enhanced stability in
aqueous systems, controlled interaction with cells, and prolonged
systemic half-life. Non-limiting examples of suitable PEGs include
those with end functionality such as methyl, hydroxyl, amine, acid
etc, functionality, and/or those with molecular weights above 300
g/mol, preferably those with hydroxyl and acid functional chains
and/or with molecular weights >750 g/mol. Particularly preferred
are hydroxyl compounds and/or those with molecular weights >1000
g/mol. Alternatively, other chemical moieties which function in the
same or similar way and which can advantageously be used in the
present invention include acrylate and methacrylate moieties
including water-soluble polymeric chains (e.g. less than 20000
g/mol), for example derived from vinyl or non vinyl monomers such
as ethylene glycol methacylate, glycerol methacrylate, vinyl
alcohol, acrylic acid, methacrylic acid, or hydroxyethyl
methacrylate.
[0033] The initiators may include groups which allow
post-functionalization of the polydendrons. Thus, whilst various
possible initiator structures and moieties have been discussed
above, an alternative to them being present within the initiator at
the start of the reaction is to incorporate them later by reaction
of the polydendron with suitable materials.
[0034] Suitable functional groups in initiators which allow
post-functionalization include thiols, hydroxyl groups, amines,
acids or isocyanates, amongst others.
[0035] For example, N-hydroxysuccinimide functionalized initiators
can be incorporated into polydendrons and post-functionalized with
materials containing amine groups.
[0036] The several means of flexibility and levels of control
provided by the present invention reside in the ability to alter
several variables including: the amount of initiator(s) relative to
vinyl polymer, the ratio between dendron initiator(s) and
non-dendron initiator(s) [or other dendron initiator(s)], the
nature and properties of the dendron initiator(s), the nature and
properties of the non-dendron initiator(s), the extent of
branching, the nature and properties of the monomer(s), the nature
and properties of the brancher(s), and the capacity of the
nanomaterials for drugs or other materials.
[0037] A further advantage of the methods and products of the
present invention is that they are compatible with the preparation
of nanomaterials which are stable and of controllable and uniform
size. Nanoprecipitation of branched vinyl polymers is disclosed in
R. A. Slater, T. O McDonald, D. J. Adams, E. R. Draper, J. V. M.
Weaver and S. P. Rannard, Soft Matter 2012, 8, 9816-9827. This
technique has been successfully used on single and mixed
initiator--carrying polydendrons of the present invention to
prepare stable nanoparticles. The nanoparticles are prepared by
self assembly during precipitation with the dispersity and size of
these nanoparticles being effectively controlled by varying the
nature of the solvents, precipitation method, concentration, and
presence of other components. Uniform or near uniform assembled
nanoparticle sizes with low polydispersities can be achieved.
Nanoparticles of uniform and controllable size are extremely useful
in the field of drug encapsulation and delivery.
[0038] The nanoparticles may for example be prepared by
precipitation of the polydendron out of solution using a solvent
which is a non-solvent for the vinyl polymer scaffold but which is
a good solvent for the dendrons or other surface groups.
[0039] This nanoprecipitation using a solvent switch might have
been expected to lead to collapse of the internal vinyl polymer
core, but self-assembly of the individual polydendron particles is
observed leading to very stable distributions of larger complex
nanoparticles with a narrow size distribution.
[0040] A preferred "non-solvent" for the vinyl polymer, i.e. medium
in which the nanoprecipitate particles are stable, is water.
[0041] By way of example, where the core is a polyHPMA-EGDMA
material and the dendrons are selected from G1 or G2 (shown in FIG.
1), then the material can be first dissolved in THF and
nanoprecipitated into water, or first dissolved in acetone and then
precipitated by adding hexane.
[0042] The characteristics of the polydendron, including the
electronic/charge and steric nature, and the nature of the solvent,
affect the way in which the material behaves in that solvent.
Without wishing to be bound by theory, the particles generally
increase in size until they reach a colloidally stable state during
the nanoprecipitation process.
[0043] As exemplified below, the present invention allows the
encapsulation and release of not only organic materials--e.g. nile
red, simulating encapsulation of a drug--but also inorganic
materials--e.g. magnetic particles. This expands the utility of the
present invention to cover further therapeutic and targeting uses.
The encapsulation of inorganic material (e.g. magnetic material,
e.g. iron oxide) in polydendrons may also be considered as a
standalone invention within this disclosure.
[0044] The branches are typically distributed statistically
throughout the connected linear polymer chains (rather than
discretely in block polymerised monofunctional vinyl monomers and
difunctional vinyl monomers). Each branch may be a glycol diester
branch, for example.
[0045] The difunctional vinyl monomer acts as a brancher (or
branching agent) and provides a branch between adjacent polymer
chains. The branching agent may have two or more vinyl groups.
[0046] The monofunctional monomer utilised for the primary chain
may comprise any carbon-carbon unsaturated compound which can be
polymerised by an addition polymerisation mechanism, for example
vinyl and allyl compounds. The monofunctional monomer may be
hydrophilic, hydrophobic, amphiphilic, anionic, cationic, neutral
or zwitterionic in nature.
[0047] The monofunctional monomer may be selected from but is not
necessarily limited to monomers such as: vinyl acids and
derivatives (including esters, amides and anhydrides), vinyl aryl
compounds, vinyl ethers, vinyl amines and derivatives (including
aryl amines), vinyl nitriles, vinyl ketones, and derivatives of the
aforementioned compounds as well as corresponding allyl variants
thereof.
[0048] Vinyl acids and derivatives thereof include: (meth)acrylic
acid, fumaric acid, maleic acid, itaconic acid and acid halides
thereof such as (meth)acryloyl chloride.
[0049] Vinyl acid esters and derivatives thereof include: C1 to C20
alkyl(meth)acrylates (linear and branched) such as for example
methyl (meth)acrylate, stearyl (meth)acrylate and 2-ethyl hexyl
(meth)acrylate; aryl(meth)acrylates such as for example benzyl
(meth)acrylate; tri(alkyloxy)silylalkyl(meth)acrylates such as
trimethoxysilylpropyl(meth)acrylate; and activated esters of
(meth)acrylic acid such as N-hydroxysuccinamido(meth)acrylate.
[0050] Vinyl aryl compounds and derivatives thereof include:
styrene, acetoxystyrene, styrene sulfonic acid, 2- and 4-vinyl
pyridine, vinyl naphthalene, vinylbenzyl chloride and vinyl benzoic
acid.
[0051] Vinyl acid anhydrides and derivatives thereof include:
maleic anhydride. Vinyl amides and derivatives thereof include:
(meth)acrylamide, N-(2-hydroxypropyl)methacrylamide, N-vinyl
pyrrolidone, N-vinyl formamide, (meth)acrylamidopropyl trimethyl
ammonium chloride, [3-((meth)acrylamido)propyl]dimethyl ammonium
chloride, 3-[N-(3-(meth)acrylamidopropyl)-N,N-dimethyl]aminopropane
sulfonate, methyl (meth)acrylamidoglycolate methyl ether and
N-isopropyl(meth)acrylamide.
[0052] Vinyl ethers and derivatives thereof include: methyl vinyl
ether.
[0053] Vinyl amines and derivatives thereof include:
dimethylaminoethyl (meth)acrylate, diethylaminoethyl
(meth)acrylate, diisopropylaminoethyl (meth)acrylate,
mono-t-butylaminoethyl (meth)acrylate,
morpholinoethyl(meth)acrylate and monomers which can be
post-reacted to form amine groups, such as N-vinyl formamide.
[0054] Vinyl aryl amines and derivatives thereof include: vinyl
aniline, 2 and 4-vinyl pyridine, N-vinyl carbazole and vinyl
imidazole.
[0055] Vinyl nitriles and derivatives thereof include:
(meth)acrylonitrile.
[0056] Vinyl ketones or aldehydes and derivatives thereof include:
acreolin.
[0057] Monomers based on styrene or those containing an aromatic
functionality such as styrene, .alpha.-methyl styrene, vinyl benzyl
chloride, vinyl naphthalene, vinyl benzoic acid, N-vinyl carbazole,
2-, 3- or 4-vinyl pyridine, vinyl aniline, acetoxy styrene, styrene
sulfonic acid, vinyl imidazole or derivatives thereof may also be
used.
[0058] Other suitable monofunctional monomers include:
hydroxyl-containing monomers and monomers which can be post-reacted
to form hydroxyl groups, acid-containing or acid-functional
monomers, zwitterionic monomers and quaternised amino monomers.
[0059] Hydroxyl-containing monomers include: vinyl hydroxyl
monomers such as hydroxyethyl (meth)acrylate, 1- and 2-hydroxy
propyl (meth)acrylate, 2-hydroxy methacrylamide, glycerol
mono(meth)acrylate and sugar mono(meth)acrylates such as glucose
mono(meth)acrylate.
[0060] Monomers which can be post-reacted to form hydroxyl groups
include: vinyl acetate, acetoxystyrene and glycidyl
(meth)acrylate.
[0061] Acid-containing or acid functional monomers include:
(meth)acrylic acid, styrene sulfonic acid, vinyl phosphonic acid,
vinyl benzoic acid, maleic acid, fumaric acid, itaconic acid,
2-(meth)acrylamido 2-ethyl propanesulfonic acid,
mono-2-((meth)acryloyloxy)ethyl succinate and ammonium sulfatoethyl
(meth)acrylate.
[0062] Zwitterionic monomers include: (meth)acryloyl
oxyethylphosphoryl choline and betaines, such as
[2-((meth)acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium
hydroxide.
[0063] Quaternised amino monomers include:
(meth)acryloyloxyethyltri-(alk/aryl)ammonium halides such as
(meth)acryloyloxyethyltrimethyl ammonium chloride.
[0064] Oligomeric, polymeric and di- or multi-functionalised
monomers may also be used, especially oligomeric or polymeric
(meth)acrylic acid esters such as mono(alk/aryl) (meth)acrylic acid
esters of polyalkyleneglycol or polydimethylsiloxane or any other
mono-vinyl or allyl adduct of a low molecular weight oligomer.
[0065] Oligomeric and polymeric monomers include: oligomeric and
polymeric (meth)acrylic acid esters such as
mono(alk/aryl)oxypolyalkyleneglycol(meth)acrylates and
mono(alk/aryl)oxypolydimethyl-siloxane(meth)acrylates. These esters
include for example: monomethoxy oligo(ethyleneglycol)
mono(meth)acrylate, monomethoxy oligo(propyleneglycol)
mono(meth)acrylate, monohydroxy oligo(ethyleneglycol)
mono(meth)acrylate, monohydroxy oligo(propyleneglycol)
mono(meth)acrylate, monomethoxy poly(ethyleneglycol)
mono(meth)acrylate, monomethoxy poly(propyleneglycol)
mono(meth)acrylate, monohydroxy poly(ethyleneglycol)
mono(meth)acrylate and monohydroxy poly(propyleneglycol)
mono(meth)acrylate.
[0066] Vinyl acetate and derivatives thereof can also be
utilised.
[0067] Further examples include: vinyl or allyl esters, amides or
ethers of pre-formed oligomers or polymers formed via ring-opening
polymerisation such as oligo(caprolactam), oligo(caprolactone),
poly(caprolactam) or poly(caprolactone), or oligomers or polymers
formed via a living polymerisation technique such as
poly(1,4-butadiene).
[0068] The corresponding allyl monomers to those listed above can
also be used where appropriate.
[0069] Specific examples of monofunctional monomers include:
amide-containing monomers such as (meth)acrylamide,
N-(2-hydroxypropyl)methacrylamide, N,N'-dimethyl(meth)acrylamide, N
and/or N'-di(alkyl or aryl)(meth)acrylamide, N-vinyl pyrrolidone,
[3-((meth)acrylamido)propyl]trimethyl ammonium chloride,
3-(dimethylamino)propyl(meth)acrylamide,
3-[N-(3-(meth)acrylamidopropyl)-N,N-dimethyl]aminopropane
sulfonate, methyl (meth)acrylamidoglycolate methyl ether and
N-isopropyl(meth)acrylamide; (meth)acrylic acid and derivatives
thereof such as (meth)acrylic acid, (meth)acryloyl chloride (or any
halide), (alkyl/aryl)(meth)acrylate; vinyl amines such as
aminoethyl (meth)acrylate, dimethylaminoethyl (meth)acrylate,
diethylaminoethyl (meth)acrylate, diisopropylaminoethyl
(meth)acrylate, mono-t-butylamino (meth)acrylate,
morpholinoethyl(meth)acrylate; vinyl aryl amines such as vinyl
aniline, vinyl pyridine, N-vinyl carbazole, vinyl imidazole, and
monomers which can be post-reacted to form amine groups, such as
vinyl formamide; vinyl aryl monomers such as styrene, vinyl benzyl
chloride, vinyl toluene, alpha-methyl styrene, styrene sulfonic
acid, vinyl naphthalene and vinyl benzoic acid; vinyl hydroxyl
monomers such as hydroxyethyl (meth)acrylate, hydroxy propyl
(meth)acrylate, glycerol mono(meth)acrylate or monomers which can
be post-functionalised into hydroxyl groups such as vinyl acetate,
acetoxy styrene and glycidyl (meth)acrylate; acid-containing
monomers such as (meth)acrylic acid, styrene sulfonic acid, vinyl
phosphonic acid, vinyl benzoic acid, maleic acid, fumaric acid,
itaconic acid, 2-(meth)acrylamido 2-ethyl propanesulfonic acid and
mono-2-((meth)acryloyloxy)ethyl succinate or acid anhydrides such
as maleic anhydride; zwitterionic monomers such as (meth)acryloyl
oxyethylphosphoryl choline and betaine-containing monomers, such as
[2-((meth)acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium
hydroxide; [0070] quaternised amino monomers such as
(meth)acryloyloxyethyltrimethyl ammonium chloride. vinyl acetate or
vinyl butanoate or derivatives thereof.
[0071] The corresponding allyl monomer, where applicable, can also
be used in each case.
[0072] Mixtures of more than one monomer may also be used to give
statistical, graft, gradient or alternating copolymers.
[0073] Some preferred monofunctional vinyl monomers include
methacrylate monomers or styrene. Some preferred hydrophobic
methacrylate monomers include 2-hydroxypropyl methacrylate (HPMA),
n-butyl methacrylate (nBuMA), tert-butyl methacrylate (tBuMA), and
oligo(ethylene glycol) methyl ether methacrylate (OEGMA). HPMA is
particularly preferred, and is readily available or synthesised as
a mixture of (predominantly) 2-hydroxypropyl methacrylate and
2-hydroxyisopropyl methacrylate. A preferred hydrophilic
methacrylate monomers is diethylaminoethyl methacrylate
(DEAEMA).
[0074] The polydendron also contains a brancher which is a
multifunctional (at least difunctional) vinyl containing
molecule.
[0075] The multifunctional monomer or brancher may comprise a
molecule containing at least two vinyl groups which may be
polymerised via addition polymerisation. The molecule may be
hydrophilic, hydrophobic, amphiphilic, neutral, cationic,
zwitterionic, oligomeric or polymeric. Such molecules are often
known as cross-linking agents in the art.
[0076] Examples include: di- or multivinyl esters, di- or
multivinyl amides, di- or multivinyl aryl compounds, di- or
multivinyl alk/aryl ethers. Typically, in the case of oligomeric or
polymeric di- or multifunctional branching agents, a linking
reaction is used to attach a polymerisable moiety to a di- or
multifunctional oligomer or polymer. The brancher may itself have
more than one branching point, such as T-shaped divinylic oligomers
or polymers. In some cases, more than one multifunctional monomer
may be used. The corresponding allyl monomers to those listed above
can also be used where appropriate.
[0077] Preferred multifunctional monomers or branchers include but
are not limited to:
divinyl aryl monomers such as divinyl benzene; (meth)acrylate
diesters such as ethylene glycol di(meth)acrylate, propyleneglycol
di(meth)acrylate and 1,3-butylenedi(meth)acrylate; polyalkylene
oxide di(meth)acrylates such as tetraethyleneglycol
di(meth)acrylate, poly(ethyleneglycol)di(meth)acrylate and
poly(propyleneglycol)di(meth)acrylate; divinyl(meth)acrylamides
such as methylene bisacrylamide; silicone-containing divinyl esters
or amides such as (meth)acryloxypropyl-terminated
poly(dimethylsiloxane); divinyl ethers such as
poly(ethyleneglycol)divinyl ether; and tetra- or tri-(meth)acrylate
esters such as pentaerythritol tetra(meth)acrylate,
trimethylolpropane tri(meth)acrylate or glucose di- to
penta(meth)acrylate.
[0078] Further examples include: vinyl or allyl esters, amides or
ethers of pre-formed oligomers or polymers formed via ring-opening
polymerisation such as oligo(caprolactam), oligo(caprolactone),
poly(caprolactam) or poly(caprolactone), or oligomers or polymers
formed via a living polymerisation technique such as oligo- or
poly(1,4-butadiene). Some preferred types of difunctional vinyl
monomers include dimethacrylate monomers, for example
ethyleneglycol dimethacrylate (EGDMA).
[0079] The molar ratio of difunctional vinyl monomer to initiator
is preferably no more than 2, more preferably no more than 1.5, and
most preferably no more than 1 if conducted under appropriate
conditions.
[0080] The amount of difunctional vinyl monomer relative to
monofunctional vinyl monomer is preferably 7.5 mol % or less, 2 mol
% or less, or 1.6 mol % or less, more preferably between 1 and 7.5
mol %, for example between 1 and 2 mol %
[0081] In a preferred embodiment, the method is a one-pot method.
In this embodiment, the reaction of monofunctional vinyl monomer,
difunctional vinyl monomer and initiators is carried out
conveniently and cost-effectively.
[0082] Preferably the method comprises preparing a mixture of the
monofunctional vinyl monomer, difunctional vinyl monomer and
initiators under suitable conditions. The mixture may contain a
catalyst (such as CuCl) or additional agents depending on the
addition polymerisation technique being used. The mixture may also
contain a ligand (such as 2,2'-bipyridine). The mixture may also
contain a chain transfer agent.
[0083] Suitable ATRP initiators include isobutyrate esters,
preferably haloisobutyrate esters, most preferably bromoisobutyrate
esters. Thus the initiator can for example have the following
general formula I:
##STR00001##
wherein X denotes a chemically addressable group and is preferably
a halide, for example Cl or Br, most preferably Br; and wherein R
is any suitable organic moiety. Where the initiator is a dendron
initiator, R is branched into a dendritic wedge and X is the
chemically addressable group at the apex of the dendritic wedge.
Whilst isobutyryl esters are convenient and effective to use in
this context, other chemistries are possible.
[0084] It of course will be understood that part of the initiator
(in this case the X group, usually bromide) is present in the
initiator but reacts during the process so that it is not
necessarily present in the product at the end of all primary
chains.
[0085] Where the initiator of general formula I is a dendron
initiator, R is a moiety which divides into two or more (preferably
two) first generation branches (preferably identical first
generation branches). Optionally each of those first generation
branches then divides into two or more (preferably two) second
generation branches (preferably identical second generation
branches). Optionally each of those second generation branches then
divides into two or more (preferably two) third generation branches
(preferably identical third generation branches). There may
analogously be further generations of branching. A dendron having
only first generation branches is known as a generation 1 dendron;
a Dendron having first and second generation branches is known as a
generation 2 dendron.
[0086] The outermost branches of the dendron (the part most likely
to end up on the surface of the polydendron) may comprise one or
more of a variety of chemical groups, for example aromatic groups
(e.g. benzene rings, e.g. of benzyloxy groups), amines (e.g.
tertiary amines), alkyl groups (e.g. alkyl chains or branched alkyl
groups e.g. tertiary butyl groups), amide groups, xanthates or
carbamates (e.g. terminating in a tertiary butyl group). These are
however merely non-limiting examples: many chemistries are
possible. One of the advantages of the present invention is that is
compatible with a wide variety of different types of dendrons and
other groups; the flexibility provided by the use of mixed
initiators is considerable. The properties can be tuned by
selecting dendrons with different chemical constituents and/or
different surface groups, for example hydrophilic or hydrophobic
groups, large or small moieties, groups of different polar or
electronic character, groups which may allow further conjugation,
etc.
[0087] Each segment may comprise one or more of an alkyl chain,
ester, carbamate, or other linking group. Again these are merely
non-limiting examples and many chemistries are possible.
[0088] Within the dendron, the structure may divide at any suitable
point, for example a carbon atom or a nitrogen atom, or a larger
moiety such as a ring. For example the structure may comprise a
N,N-bis-substituted amino component, e.g. esters of
1-[N,N-bis-substituted amino]-2-propanol.
[0089] Some specific and non-limiting examples of possible dendrons
will now be described.
[0090] A first class of possible dendrons include those having
benzyloxy surface groups. For example the surface group may have
the following structure:
##STR00002##
[0091] Optionally two of these moieties may be linked via carbamate
chains to an amide branching point.
[0092] Examples in this class of dendrons include the G1 and G2
structures shown in FIG. 1.
[0093] A second class of possible dendrons include those having
tertiary amine surface groups, for example where the end amines are
dimethyl substituted. Optionally the branching may occur at
tertiary amine centres and the segments may contain ester
linkages.
[0094] Examples in this class of dendrons, and a suitable component
thereof, are shown in FIG. 2.
[0095] A third class of possible dendrons include those having
carbamate surface functionality, for example tertiary butyl
carbamates, and optionally carbamate functionality within the
segment(s).
[0096] Examples in this class of dendrons are shown in FIG. 3.
[0097] A fourth class of possible dendrons include those having
xanthate functionality, optionally with branches comprising
esters.
[0098] Examples in this class of dendrons, and a suitable component
thereof, are shown in FIG. 4.
[0099] The dendrons may be prepared by known chemical techniques.
Some possible methods of preparation include those described
below.
[0100] The present invention will now be described in further
non-limiting detail and with reference to the Examples and Figures
in which:
[0101] FIGS. 1 to 4 show some examples of dendron initiators and
components thereof used in the present invention;
[0102] FIG. 5 shows, schematically, structural differences between
dendrimers and polydendrons;
[0103] FIGS. 6 and 7 show MTT assays of Caco-2 cells following
incubation with aqueous Nile Red and polydendrons;
[0104] FIGS. 8 and 9 show ATP assays of Caco-2 cells following
incubation with aqueous Nile Red and polydendrons;
[0105] FIG. 10 shows results in relation to transcellular
permeability of selected Nile Red polydendron materials across
Caco-2 cell monolayers
[0106] FIG. 11 shows, schematically, how using different dendron:
polyethylene glycol initiator ratios can result in a spectrum of
hydrophobicity, amphiphilicity and hydrophilicity;
[0107] FIG. 12 is a photograph, corresponding to FIG. 11, and
illustrates how using different dendron:polyethylene glycol
initiator ratios can affect the response of encapsulated Nile
Red;
[0108] FIG. 13 shows, schematically, one method of
nanoprecipitation of polydendrons;
[0109] FIGS. 14a and 14b are SEM images of polydendron
nanoprecipitates.
[0110] The experimental details below relate to: preparative
procedures for various dendron and non-dendron initiators used in
the present invention, including initiators containing polyethylene
glycol (PEG) and sugar moieties; preparative procedures and
properties of various polydendrons showing how hydrophilic or
hydrophobic properties can be tailored; nanoprecipitation methods
and results; encapsulation experiments showing how molecules can be
encapsulated and showing the effect of tailoring the encapsulation
environment, as a model for drug encapsulation; cytotoxicity
analysis using MTT and ATP assays in respect of Caco-2 cells;
transcellular permeability of polydendrons carrying Nile Red (to
model drug transfer across the intestinal epithelium); and
encapsulation of inorganic material (e.g. magnetic particles).
[0111] Very positive results were obtained with regard to
cytotoxicity and in the drug transport model. The experiments below
show in particular that a material which would otherwise not pass
effectively from gut to blood can be carried over by using
polydendrons of the present invention.
[0112] Whereas a representation of an ideal dendrimer structure is
shown in FIG. 5a, the present invention is concerned with
polydendrons which have dendrons and a polymer core as represented
in Figure Sc, constituent parts of which include dendrons attached
to polymer chains as represented in FIG. 5b. The polydendron
represented in FIG. 5c has several dendrons of the same type;
however the focus of the present invention is on polydendrons which
have a branched polymer core and which carry not only one type of
dendron moiety but also at least one further moiety, whether that
be a dendron moiety or a non-dendron moiety.
[0113] In other words the polydendrons can be prepared by using
mixed initiators, to end up with polydendron structures as
represented for example in FIG. 11. At the far left of FIG. 11 is
represented a hydrophilic polydendron made using 100% dendron
initiator, at the far right of FIG. 11 is represented a hydrophobic
material made using 100% PEG. The
hydrophobicity/amphiphilicity/hydrophilicity can be tuned by
varying the relative amounts of the different initiators.
[0114] FIG. 12 is a photograph of vials containing the seven
different types of polydendron shown schematically in FIG. 11 (i.e.
100% dendron initiator with 0% PEG initiator on the left, through
to 0% dendron initiator with 100% PEG initiator on the right)
carrying Nile Red. In the original photograph, the darkest pink
colour can be seen on the left, lighter pinks in the middle vials,
and a very pale pink on the right, thereby showing that the
hydrophobicity can be tuned in a discernible and controllable
manner.
[0115] Whilst the present invention is primarily focused on the use
of mixed initiators to prepare polydendrons, and the products
themselves, nevertheless the present invention also covers the
corresponding methods and products wherein only one type of
initiator is used, in other words where one of the dendron
initiators disclosed herein is used.
[0116] Novel products, components thereof, intermediates, methods
or method steps, disclosed herein, also fall within the scope of
the present invention
EXAMPLES
1. Initiator Syntheses
[0117] 1.1 Protected Sugar Initiator
##STR00003##
[0118] Lactose (4 g, 11.7 mmol) was weighed into a 100 mL round
bottom flask equipped with a magnetic stirrer and dry N.sub.2
inlet. The flask was purged with nitrogen for 15 minutes. Acetic
anhydride (30 mL) and Iodine (208 mg, 1.58 mmol) were added,
instantly forming a brown coloured solution. Within 10 minutes the
flask began to warm due to onset of acetylation. The solution was
stirred overnight at room temperature under a positive flow of
nitrogen. The solution was transferred to a 250 mL separating
funnel containing dichloromethane (50 mL), sodium thiosulfate
solution (30 mL) and crushed ice, and the product was extracted
into the organic layer. The aqueous layer was further extracted
with dichloromethane (2.times.50 mL). The organic phases were
collected and washed with saturated sodium carbonate solution until
neutral. The organic phase was collected, dried over anhydrous
MgSO.sub.4, and concentrated in vacuo to give a white solid.
##STR00004##
[0119] Lactose octa-acetate (5.1 g, 7.52 mmol) was weighed into a
250 mL, round bottom flask equipped with a magnetic stirrer, and
was dissolved in tetrahydrofuran (100 mL). Ethylene diamine (0.6
mL, 9.02 mmol) was added to the flask, followed by the slow
addition of acetic acid (0.6 mL, 10.5 mmol), to give a white
coloured turbid solution. A gas was evolved and the flask warmed
slightly upon addition of the acid. The flask was lightly sealed
with a rubber septum cap, and stirred overnight at room
temperature, to give a cream coloured mixture. Distilled water (50
mL) was added to the flask, whereby the precipitate dissolved,
leaving a slightly yellow coloured solution. The solution was
transferred to a 500 mL separating funnel containing
dichloromethane (100 mL), and the product was extracted into the
organic solvent. A further extraction of the aqueous layer was
performed with dichloromethane (50 mL). The organic layers were
combined, washed with hydrochloric acid (80 mL, 2M), saturated
sodium bicarbonate solution (80 mL) and distilled water (80 mL).
The organic layer was dried over anhydrous MgSO.sub.4, filtered and
concentrated in vacuo. The crude product was purified by flash
column chromatography (silica, eluent hexane/acetone, 60/40) to
give a white solid.
##STR00005##
[0120] Lactose septa-acetate (3 g, 4.71 mmol) was added to a 50 mL
round bottom flask equipped with a magnetic stirrer and dry N.sub.2
inlet. The flask was then purged with nitrogen for 10 minutes.
Anhydrous tetrahydrofuran (8 mL) was added to the flask, and
N.sub.2 was bubbled through the mixture for a further 10 minutes.
Triethylamine (0.99 mL, 7.07 mmol) was added to a vial, diluted
with tetrahydrofuran (2 mL), and then transferred to the reaction
flask drop-wise. Following this, 2-bromoisobutyryl bromide (0.87
mL, 7.07 mmol) was added to a vial, diluted with tetrahydrofuran (2
mL) and transferred to the reaction flask drop-wise. Reaction
mixture was left to stir overnight at room temperature under a
positive flow of nitrogen. This gave a white coloured turbid
mixture. The mixture was filtered by gravity filtration, the
precipitate washed with tetrahydrofuran, and the solution
concentrated in vacuo. The crude product was purified by flash
column chromatography (silica, eluent hexane/ethyl acetate, 95/5)
to give a white solid.
[0121] 1.2 PEG Initiators
[0122] 1.2.1 750-PEG Initiator
##STR00006##
[0123] Monomethoxy poly(ethylene glycol) (Mw.apprxeq.750
gmol.sup.-1) (23.0 g, 30.7 mmol) was dissolved in warm THF
(.about.40.degree. C.), and the reaction was degassed with dry
N.sub.2. DMAP (37.5 mg, 0.3 mmol) and TEA (7.48 ml, 53.7 mmol) were
added and the reaction was cooled to 0.degree. C. in an ice bath.
.alpha.-bromo isobutyryl bromide (5.69 ml, 46.0 mmol) was added
dropwise over 30 minutes and a white precipitate appeared
immediately; the EtNH.sup.+Br.sup.- salt. After 24 hours the
precipitate was filtered, THF removed in vacuo and the resulting
crude product was precipitated from acetone into petroleum ether
(30-40.degree. C.) twice (72%). .sup.1H NMR (400 MHz, D.sub.2O)
.delta. ppm 4.31 (m, 2H), 3.77 (m, 2H), 3.70-3.59 (m, 60H), 3.55
(m, 2H), 3.31 (s, 3H) and 1.89 (s, 6H).
[0124] 1.2.2 2K-PEG Initiator
##STR00007##
[0125] Monomethoxy poly(ethylene glycol) (Mw.apprxeq.2000
gmol.sup.-1) (20.5 g, 10.25 mmol) was dissolved in warm THF
(.about.40.degree. C.), and the reaction was degassed with dry
N.sub.2. DMAP (12.5 mg, 0.1 mmol) and TEA (3.14 ml, 22.5 mmol) were
added and the reaction was cooled to 0.degree. C. in an ice bath.
.alpha.-bromo isobutyryl bromide (2.53 ml, 20.5 mmol) was added
dropwise over 20 minutes and a white precipitate appeared
immediately; the Et.sub.3NH.sup.+Br.sup.- salt. After 24 hours the
precipitate was filtered, THF removed in vacuo and the resulting
crude product was precipitated from acetone into petroleum ether
(30-40.degree. C.) twice (89%). .sup.1H NMR (400 MHz, D.sub.2O)
.delta. ppm 4.34 (m, 2H), 3.80-3.59 (m, 186H), 3.35 (s, 3H) and
1.93 (s, 6H).
[0126] 1.3 G0 (Non-Dendron) Initiators
[0127] 1.3.1 G0 Tertiary Amine Functional Initiator
##STR00008##
[0128] 1-dimethylamino-2-propanol (1.1207 g, 10.86 mmol, 1 eq.),
TEA (1.5390 g, 15.2 mmol, 1.4 eq.) and DMAP (132.7 mg, 1.086 mmol,
0.1 eq.) were added to a 250 mL 2 necked round-bottomed flask
containing DCM (160 mL). The flask was deoxygenated under a
positive N.sub.2 purge for 10 minutes. .alpha.-bromoisobutyryl
bromide (2.622 g, 1.4 mL, 11.4 mmol, 1.05 eq.) was added drop wise
while the solution was stirring in an ice bath under a positive
flow of N.sub.2. The reaction mixture was allowed to warm to room
temperature and left stirring overnight. The organic phase was
washed with saturated sodium hydrogen carbonate (NaHCO.sub.3)
solution (3.times.30 mL). The solution was dried with anhydrous
Na.sub.2SO.sub.4. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.27
(d, 3H), 1.89 (m, 6H), 2.17-2.55 (m, 8H), 5.07 (m, 1H). m/z (ES MS)
252 [M+H].sup.+.
[0129] 1.4 G1, G2 Dendron Initiators
[0130] 1.4.1 G1-Aromatic Dendron Initiator (G1 DBOP Br)
##STR00009##
[0131] 1,3-Dibenzyloxy-2-propanol, 1, (9.80 g, 36.0 mmol) was
weighed into a 2-neck round bottom flask which was equipped with
magnetic stirrer and dry N.sub.2 inlet. Dichloromethane (DCM) (100
ml) was added followed by 4-(dimethylamino)pyridine (DMAP) (0.44 g,
3.6 mmol) and triethylamine (TEA) (7.53 ml, 54.0 mmol). The
reaction was cooled to 0.degree. C. in an ice-bath and
.alpha.-bromoisobutyryl bromide (5.34 ml, 43.2 mmol) was added
dropwise over 20 minutes. After complete addition the reaction was
warmed to room temperature and left stirring overnight. Reaction
could be observed by the formation of a white precipitate. After 24
hours the precipitate was removed by filtration, the resulting
crude reaction medium was washed first with a saturated solution of
NaHCO.sub.3 (3.times.100 ml) followed by distilled water
(3.times.100 ml). The organic layer was dried over Na.sub.2SO.sub.4
and concentrated in vacuo to give a pale yellow oil (81%). Found,
C, 59.55; H, 6.02%. C.sub.21H.sub.25BrO.sub.4 requires, C, 59.86;
H, 5.98; Br, 18.96; O, 15.19%. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. ppm 7.35-7.20 (m, 10H), 5.26 (m, 1H), 4.55 (m, 4H), 3.69
(d, 4H), 1.93 (s, 6H). .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
ppm 171.2, 138.0, 128.4, 127.7, 127.6, 73.3, 68.5, 55.8, 30.7. m/z
(ES MS) 443.1 [M+Na].sup.+, 461.1 [M+K].sup.+, m/z required 420.1
[M].sup.+.
##STR00010## ##STR00011##
[0132] 1,1'-Carbonyldiimidazole (CDI) (9.73 g, 60.0 mmol) was
weighed into a 2-neck round bottom flask and equipped with magnetic
stirring, condenser and dry N.sub.2 inlet. Anhydrous toluene (100
ml) was added, followed by KOH (0.34 g, 6.0 mmol) and 1 (12.35 ml,
50.0 mmol). The reaction was heated to 60.degree. C. for 6 hours.
Toluene was removed in vacuo, the crude mixture was dissolved in
DCM (50 ml) and washed with distilled water (3.times.50 mil). The
organic layer was dried over Na.sub.2SO.sub.4 and concentrated in
vacuo to give 3, a pale yellow oil (97%). Found C, 68.64; H, 6.10;
N, 7.85%. C.sub.21H.sub.22N.sub.2O.sub.4 requires C, 68.84; I,
6.05; N, 7.65; O, 17.47%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
ppm 8.11 (s, 1H), 7.41 (s, 1H), 7.33-7.23 (m, 10H), 7.06 (s, 1H),
5.36 (qn, 1H), 4.53 (m, 4H), 3.75 (m, 4H). .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. ppm 148.3, 137.5, 137.2, 130.6, 128.4, 127.9,
127.6, 117.2, 76.1, 73.3, 68.1. m/z (ES MS) 367.2 [M+H], 389.2
[M+Na]+, 405.1 [M+K].sup.+, m/z required 366.2 [M].sup.+.
[0133] 3 (16.84 g, 46.0 mmol) was weighed into a 2-neck round
bottom flask which was equipped with magnetic stirring, condenser
and dry N.sub.2 inlet. Anhydrous toluene (120 ml) was added
followed by diethylenetriamine (DETA) (2.48 ml, 23.0 mmol). The
reaction was heated to 60.degree. C. for 48 hours. Toluene was
removed in vacuo, the resulting crude mixture was dissolved in DCM
(100 ml) and washed with distilled water (3.times.100 ml). The
organic layer was dried over Na.sub.2SO.sub.4 and concentrated in
vacuo to give 4, a yellow oil (93%). Found C, 68.50; H, 7.13; N,
6.00%. C.sub.40H.sub.49N.sub.3O requires, C, 68.65; H, 7.06; N,
6.00; O, 18.29%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm
7.27-7.16 (m, 20H), 5.23 (s, br, NH), 5.03 (qn, 2H), 4.44 (m, 8H),
3.57 (d, 8H), 3.12 (m, 4H), 2.58 (m, 4H). .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. ppm 156.6, 138.4, 128.8, 128.1, 73.7, 72.1,
69.4, 49.0, 41.2. m/z (ES MS) 700.4 [M+H].sup.+, 722.3 [M+Na],
738.3 [M+K].sup.+, m/z required 699.4 [M].sup.+.
[0134] 4 (15.01 g, 21.4 mmol) was weighed into a 2-neck round
bottom flask, equipped with magnetic stirrer, condenser and dry
N.sub.2 inlet. Anhydrous toluene (90 ml) was added followed by
dropwise addition of .alpha.-butyrolactone (2.62 ml, 32.2 mmol).
The reaction was heated at reflux for 16 hours. Toluene was removed
in vacuo, the resulting crude mixture was dissolved in DCM (50 ml)
and washed with distilled water (3.times.50 ml). The organic layer
was dried over Na.sub.2SO.sub.4 and concentrated in vacuo to give a
yellow oil. The crude product was purified by silica gel column
chromatography with a mobile phase gradient of DCM:MeOH
(100:0-95:5-90:10) to give 5, a pale yellow oil (45%). Found C,
65.35; H, 6.72; N, 5.10%. C.sub.44H.sub.55N.sub.3O.sub.10 requires,
C, 67.24; H, 7.05; N, 5.35; O, 20.36%. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. ppm 7.34-7.25 (m, 20H), 5.35 (br, NH), 5.31
(br, NH), 5.11 (m, 2H), 4.50 (m, 8H), 4.14 (s, 1H), 3.62 (m, 8H),
3.46-3.18 (m, br, 8H), 2.45-2.22 (m, 2H), 1.18-1.05 (m, 3H).
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. ppm 174.4, 156.8, 156.6,
138.4, 138.3, 128.8, 128.1, 128.0, 73.7, 73.6, 72.6, 72.4, 69.5,
69.3, 65.1, 48.5, 46.5, 41.2, 40.3, 39.9, 22.9. m/z (ES MS) 808.4
[M+Na].sup.+, m/z required 785.4 [M].sup.+.
[0135] 5 (9.31 g, 11.85 mmol) was dissolved in DCM (100 ml) and
transferred to a round bottom flask which was equipped with
magnetic stirring and a dry N.sub.2 inlet. DMAP (0.14 g, 1.19
mmol), TEA (3.30 ml, 23.7 mmol) were added and the reaction mixture
was cooled to 0.degree. C. in an ice bath followed by dropwise
addition of .alpha.-bromoisobutyryl bromide (2.19 ml, 17.78 mmol).
The reaction was warmed to room temperature for 24 hours. A colour
change from pale orange to a dark orange/brown colour was observed
over time. No precipitate was observed, the crude reaction mixture
was washed with a saturated NaHCO.sub.3 solution (3.times.100 ml)
and distilled water (3.times.100 ml). The organic layer was dried
over Na.sub.2SO.sub.4 and concentrated in vacuo to give 6, an
orange oil (81%). Found C, 59.50; H, 6.31; N, 4.39%.
C.sub.48H.sub.60BrN.sub.3O.sub.11 requires, C, 61.67; H, 6.47; Br,
8.55; N, 4.49; O, 18.82%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
ppm 7.35-7.23 (m, 20H), 5.33 (s, br, NH), 5.10 (m, 2H), 4.52 (m,
8H), 3.71-3.53 (s, 8H), 3.52-3.12 (m, br, 8H), 2.76 (d of d, 1H),
2.47 (d of d, 1H), 1.87 (s, 6H), 1.29 (d, 3H). .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. ppm 192.5, 170.8, 156.3, 156.1, 137.9,
134.5, 128.4, 127.7, 127.6, 73.2, 73.1, 72.2, 71.8, 70.2, 69.1,
69.0, 68.8, 56.1, 48.3, 46.3, 39.6, 39.4, 38.9, 30.8, 30.7, 30.6,
19.7. m/z (ES MS) 958.3 [M+Na]+, 974.3 [M+K].sup.+, m/z required
933.3 [M].sup.+.
[0136] 1.4.3 Alternative G2 DBOP Br Synthesis
##STR00012##
[0137] 3 (14.03 g, 38.3 mmol) was added to a 2-neck round bottom
flask, which was equipped with magnetic stirring, condenser and a
N.sub.2 inlet. Anhydrous toluene (100 ml) was added and the
reaction was heated to 60.degree. C. The AB.sub.2 brancher (3.627
g, 19.2 mmol) was dissolved in anhydrous toluene (5 ml) was added
dropwise. After 18 hours the reaction was stopped, the toluene
removed in vacuo, the crude mixture was dissolved in
dichloromethane (100 ml) and washed with water (3.times.100 ml).
The organic phase was dried over Na.sub.2SO.sub.4 the solvent
removed in vacuo and the resulting yellow oil was dried further
under high vacuum to give 7, as a pale yellow oil, (94%). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.33-7.23 (m, 20H), 5.30 (s,
br, NH), 5.09 (m, 2H), 4.51 (m, 8H), 3.73 (m, 1H), 3.64 (d, 8H),
3.16 (m, 4H), 2.53 (m, 2H), 2.32 (m, 2H), 2.24 (m, 2H), 1.59 (m,
4H), 1.06 (d, 3H). m/z (ES MS) 786.4 [M+H].sup.+, 808.4
[M+Na].sup.+, m/z required 785.43 [M].sup.+.
[0138] 7, (13.381 g, 17.0 mmol) was dissolved in DCM (100 ml) and
bubbled with N.sub.2 for 20 minutes. 4-(Dimethylamino)pyridine
(DMAP) (21 mg, 0.17 mmol) and triethylamine (TEA) (3.56 ml, 26.0
mmol) were added and the reaction vessel was cooled to 0.degree. C.
.alpha.-Bromoisobutyryl bromide (2.53 ml, 20.0 mmol) was added
dropwise, then the reaction was warmed to room temperature for 24
hours. The organic phase was washed with a saturated solution of
NaHCO.sub.3 (3.times.150 ml) and distilled water (3.times.150 ml),
dried over Na.sub.2SO.sub.4 and the solvent removed in vacuo to
give an orange oil as the crude product. This was purified by
column chromatography with a silica stationary phase and mobile
phase of ethyl acetate:hcxane (4:1), to give 8 a yellow oil, (73%).
Found C, 63.24; H, 6.88; N, 4.44%.
C.sub.49H.sub.64BrN.sub.3O.sub.10 requires, C, 62.95; H, 6.90; N,
4.49%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.33-7.24 (m,
20H), 5.36 (s, br, NH), 5.09 (m, 2H), 5.03 (m, 1H), 4.51 (m, 8H),
3.64 (d, 8H), 3.16 (m, 4H), 2.64-2.35 (m, 6H), 1.89 (s, 6H), 1.60
(m, 4H), 1.22 (d, 3H). .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
ppm 171.2, 156.0, 138.1, 128.3, 127.60, 127.62, 73.2, 71.6, 70.4,
68.9, 59.1, 56.1, 52.2, 39.4, 30.6, 30.7, 27.2, 18.0. m/z (ES MS)
936.4 [M+H].sup.+, 959.4 [M+Na].sup.+, m/z required 935.4
[M].sup.+.
[0139] 1.4.4 G1, G2 tBOC Dendron Initiator Synthesis (Inc. AB.sub.2
Synthesis)
##STR00013##
[0140] Synthesis of 18--CDI (39.137 g, 0.241 mol) was added to an
oven-dried 500 mL 2-neck RBF fitted with a reflux condenser,
magnetic stirrer and a dry N.sub.2 inlet. Dry toluene (350 mL) was
added and the flask was purged with N.sub.2 for 10 minutes. The
solution was stirred at 60.degree. C. and 17 (t-Butanol) (35.7 g,
46 mL, 0.483 mol) was added via a warm syringe. The mixture was
left stirring at 60.degree. C. for 6 hours under a positive flow of
nitrogen. Following this, BAPA (16.077 g, 17.14 mL, 0.121 mol) was
added dropwise. The reaction was left stirring for a further 18
hours at 60.degree. C. under a positive flow of nitrogen, and then
allowed to cool to room temperature. The pale yellow solution was
filtered to remove any solid imidazole, and concentrated in vacuo.
The remaining oil was dissolved in dichloromethane (250 mL) washed
with distilled water (3.times.250 mL) and finally a saturated brine
solution (150 mL). The organic layer was dried with anhydrous
Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give 18 as
a white solid powder. 38 g, (95%). Found C, 57.84; H, 10.45; N,
12.91%. C.sub.16H.sub.33N.sub.3O.sub.4 requires, C, 57.98; H,
10.04; N, 12.68%. .sup.1H NMR (400 MHz, CDCl.sub.3) 5.19 (s, br,
NH--disappears on addition of D.sub.2O), 3.21 (t, 4H), 2.65 (t,
4H), 1.65 (q, 4H), 1.44 (s, 18H).sup.3C NMR (100 MHz, CDCl.sub.3)
156.48, 79.34, 47.77, 39.29, 30.11, 28.79. m/z (ES MS) 332.3
[M+H].sup.+
[0141] Synthesis of 19--18 (20 g, 0.06 mol) was added to a 500 mL
2-necked RBF fitted with a reflux condenser, magnetic stirrer and a
dry N.sub.2 inlet. The flask was degassed with dry nitrogen for 10
minutes, and dissolved in dry ethanol (200 mL), Whilst stirring,
and maintaining the temperature at 30.degree. C., propylene oxide
(10.51 g, 11.21 mL, 0.181 mol) was added dropwise over a period of
10 minutes. Under a positive flow of dry N.sub.2, the reaction was
left stirring at 30.degree. C. for 18 hours. After this time, the
solvent and excess propylene oxide were removed in vacuo. The crude
product was purified by liquid chromatography on silica gel,
eluting with EtOAc:MeOH, 4:1, the solvent removed in vacuo to give
19 as a pale yellow viscous oil. 19.90 g, (85%). Found C, 58.50; H,
10.23; N, 10.82%. C.sub.19H.sub.39N.sub.3O.sub.5 requires, C,
58.58; H, 10.09; N, 10.79%. .sup.1H NMR (400 MHz, CDCl.sub.3) 4.93
(s, br, NH), 3.76 (m, I H), 3.15 (m, 4H), 2.61-2.88 (m, 6H), 1.62
(m, 4H), 1.44 (s, 18H), 1.11 (d, 3H). .sup.13C NMR (100 MHz,
CDCl.sub.3) 156.08, 79.18, 63.45, 62.55, 51.77, 38.75, 27.48,
20.14. m/z (ES MS) 390.3 [M+H].sup.+
[0142] Synthesis of 20 (Part 1)--In a IL RBF, G1-OH (33.70 g) was
dissolved in ethyl acetate (330 mL) and concentrated HCl (35.03 g,
30 mL, d=1.18 36% active) was added very slowly. CO.sub.2 began to
evolve. The reaction vessel was left open and stirring for 6 hours.
.sup.1H NMR (D.sub.2O) confirmed complete decarboxylation.
Synthesis of 20 (Part 2)--After removal of ethyl acetate, the crude
oil was dissolved in 4M NaOH (300 mL), and then reduced down by
half (approx.) on the rotary evaporator (60.degree. C.). Following
this, the oily mixture was extracted twice with CHCl.sub.3 (300
mL). The organic layers were then combined, dried with anhydrous
Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give the
product as a pale yellow oil (15.27 g, 94% yield) NMR (400 MHz,
CDCl.sub.3) 3.79 (m, 1H), 2.68-2.40 (ddd, 2H), 2.31 (m, 4H), 1.89
(s, br, OH), 1.60 (m, 4H), 1.11 (d, 3H). .sup.13C NMR (100 MHz,
CDCl.sub.3) 63.95, 62.56, 52.10, 40.31, 30.80, 20.03
Preparation of t-BOC G2 Dendron, 21
##STR00014##
[0143] Synthesis of 21--19 (5 g, 12.8 mmol) was added to a 250 mL 3
necked round bottom flask containing dry toluene (60 mL), which was
fitted with a reflux condenser, magnetic stirrer and a dry N.sub.2
inlet. The flask was purged with N.sub.2 for 10 minutes. The
solution was stirred at room temperature and CDI (2.29 g, 14.1
mmole) was added via a powder addition funnel. The mixture was
heated to 60.degree. C. with stirring for 6 hours. 20 (0.91 mL, 6.4
mmole) was added dropwise whilst the solution was stirring and the
temperature was maintained at 60.degree. C. The reaction was left
overnight stirring for a further 12 hours at 60.degree. C., and
then allowed to cool to room temperature. The clear solution was
filtered to remove any solid imidazole, and concentrated in vacuo.
The crude product was purified by liquid chromatography, silica
gel, eluting with EtOAc:MeOH, 5:1, the solvent removed in vacuo to
give 21 as a pale yellow viscous oil (60%). Found C, 57.46; H,
9.83; N, 12.17%. C.sub.19H.sub.39N.sub.3O.sub.5 requires, C, 57.68;
H, 9.58; N, 12.35%. .sup.1H NMR (400 MHz, CDCl.sub.3) 4.92 (m, br,
2H), 3.74 (m, 1H), 3.35-2.93 (m, 12H), 2.73-2.14 (m, 18H), 1.62 (m,
12H), 1.44 (s, 36H), 1.20 (m, 6H), 1.10 (d, 3H) .sup.13C NMR (100
MHz, CDCl.sub.3) 156.76, 156.15, 78.91, 67.58, 63.51, 62.46, 59.36,
52.33, 51.75, 38.94, 28.50, 27.37, 20.13, 18.82, 14.20. (ES MS)
1020.7 [M+H].sup.+, 1042.7 [M+Na].
Synthesis of t-BOC Initiators 22 and 23
##STR00015##
[0144] General Procedure for Focal Point Modification to ATRP
Initiator by Acid Bromide
[0145] 19 or 20 was added to a 50 mL round bottom flask, which was
equipped with a magnetic stirrer and purged with dry N.sub.2 for 10
minutes. Following this, dichloromethane (40 mL), DMAP (0.2 eqv.)
and TEA (2 eqv.) were also added. The round bottom flask was then
purged again with dry N.sub.2, and placed into an ice bath.
Dropwise, over a period of 10 minutes 2-Bromoisobutyryl bromide
(1.1 eqv.) was added. The reaction was removed from the ice bath
after 30 minutes and left for 24 hours at room temperature. A
colour change from clear to yellow/orange was noted for all
reactions. After this time, the solution was filtered, washed with
distilled water (3.times.40 mL), washed with a saturated brine
solution (40 mL) and the organic layer dried using anhydrous
Na.sub.2SO.sub.4. The solvent was removed in vacuo, and the crude
product purified by column chromatography
[0146] Synthesis of 22--19, Bromoisobutyryl bromide (1.1 eqv.),
DMAP (0.2 eqv) and TEA (2 eqv) were allowed to react according to
the general esterification procedure above in 100 mL of dry
CH.sub.2Cl.sub.2 for 24 h. The crude product was purified by liquid
chromatography on silica gel, eluting with 95/5 DCM/MeOH increasing
to 90/10 DCM/MeOH to give 22 as a light yellow/brown viscous oil.
(77%) .sup.1H NMR (400 MHz, CDCl.sub.3) 5.06 (s, br, NH), 3.15 (m,
4H), 2.68-2.35 (m, 6H), 1.93 (s, 6H), 1.61 (q, 4H), 1.43 (s, 18H),
1.25 (d, 3H) .sup.13C NMR (100 MHz, CDCl.sub.3) 171.81, 156.05,
79.57, 70.78, 59.62, 56.36, 38.65, 31.14, 30.17, 27.36, 18.26. m/z
(ES MS) 510.2 [M+H], 534.2 [M+Na], 550.2 [M+K].sup.+
[0147] Synthesis of 23--20, Bromoisobutyryl bromide (1.1 eqv.),
DMAP (0.2 eqv) and TEA (2 eqv) were allowed to react according to
the general esterification procedure above in 100 mL of dry
CH.sub.2Cl.sub.2 for 24 h. The crude product was purified by liquid
chromatography on silica gel, eluting with 85:15 CCl.sub.3/MeOH to
give 23 as a brown viscous oil. (54%) .sup.1H NMR (400 MHz,
CDCl.sub.3) 4.92 (m, br, 2H), 3.63 (m, 1H), 3.37-2.94 (m, 12H),
2.77-2.12 (m, 18H), 1.91 (s, 6H), 1.62 (m, 121H), 1.44 (s, 36H),
1.20 (m, 9H) m/z (ES MS) 1168.7 [M+H].sup.+, 1192.7 [M+Na], 1208.7
[M+K]
[0148] 1.4.5 G1 Xanthate Dendron Initiator Synthesis (Xant-G1)
##STR00016## ##STR00017##
[0149] Synthesis of Xant b (scheme 5)--
[0150] Potassium ethyl xanthogenate (40.1 g, 250.2 mmol) was
transferred to a 500 mL two-necked round-bottomed flask, equipped
with a magnetic stirrer bar, dropping funnel and septa cap with
outlet. Acetone (150 mL) was added to the flask. 3-Bromopropionic
acid (32.4 g, 211.8 mmol) was dissolved in acetone (80 mL) and
transferred to dropping funnel. The acid was added to the flask
dropwise with stirring. Once added, the reaction was left stirring
at room temperature overnight. The initially yellow solid turns
white as the reaction proceeds. The white solid is then filtered
off and the solvent removed on the rotary evaporator. The resulting
solid was dissolved in DCM (300 mL) and washed (1.times.200 mL
distilled water and 2.times.200 mL brine). The organic layer was
dried over MgSO.sub.4, and the solid filtered off. The solvent was
removed and placed in a vacuum oven to remove any residual solvent.
Yield 59%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 1.42 (t, 3H),
2.85 (t, 2H), 3.38 (t, 2H), 4.63 (q, 2H)
[0151] Synthesis of Xant c (scheme 5)--
[0152] Xanthate carboxylic acid, Xant b in (scheme 5) (15.0 g, 77.2
mmol) was transferred to a 250 mL round-bottomed flask, equipped
with a magnetic stirrer bar and septa cap containing outlet. DCM
(100 mL) was added. 5 drops of DMF was added. Oxalyl chloride (19.6
g, 154.4 mmol) was added dropwise via syringe with stirring. The
reaction was left stirring for 2 hours. The reaction mixture
changes from clear to a transparent orange as the reaction
proceeds. The solvent was removed and washed twice with chloroform
to remove any residual oxalyl chloride. Resulting viscous orange
oil used as obtained. Yield quantitative. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta.: 1.42 (t, 3H), 3.38 (m, 4H), 4.63 (q, 2H).
[0153] Synthesis of Xant d (scheme 5)--
[0154] Bis-MPA (4.1 g, 30.9 mmol), TEA (12.9 mL, 101.2 mmol) and
DMAP (188.6 mg, 1.6 mmol) were transferred to a 250 mL two-necked)
round-bottomed flask equipped with a magnetic stirrer bar, dropping
funnel and septa cap containing outlet. The flask was then
deoxygenated using nitrogen. Dry DCM (60 mL) was added via syringe
under nitrogen. Xanthate acid chloride, Xant c in (scheme 5) . . .
(16.4 g, 77.2 mmol) was degassed with nitrogen inside the sealed
dropping funnel. Dry DCM (10 mL) was added to dissolve the acid
chloride. The xanthate acid chloride was added dropwise and the
reaction was left stirring under nitrogen overnight. The resulting
solution was washed (1.times.200 mL distilled water and 2.times.200
mL brine). The organic layer was dried over MgSO.sub.4, and the
solid filtered off. The solvent was reduced and the product was run
through an automated flash column with a starting eluent of 95:5
hexane: ethyl acetate increasing to 20:80. Product fractions
collected and solvent removed. The product was further washed with
chloroform to remove residual ethyl acetate, and solvent removed
again. Resulting oily product was placed in vacuum oven to remove
any residual solvent. Yield (35%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta.: 1.30 (s, 3H), 1.42 (t, 6H), 2.80 (t, 4H), 3.37
(t, 4H), 4.30 (m, 4H), 4.65 (q, 4H).
[0155] Synthesis of Xant e (scheme 5)--
[0156] Xant d (scheme 5) (4.8 g, 9.9 mmol) was transferred to a 100
mL round-bottomed flask equipped with a magnetic stirrer bar and
septa cap containing outlet. DCM (30 mL) was added. 5 drops of DMF
were added. Oxalyl chloride (2.5 g, 19.8 mmol) was added dropwise
via syringe. The reaction was left stirring for 3 hours. The
solution changed from pale yellow to dark orange as the reaction
proceeds. The solvent was removed and the resulting oil was washed
twice with chloroform to remove any residual oxalyl chloride. The
product was in the form of viscous brown oil. Yield quantitative.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 1.42 (m, 9H), 2.80 (t,
4H), 3.38 (t, 4H), 4.35 (m, 4H), 4.65 (q, 4H).
[0157] Synthesis of Xant-G1 (scheme 5)--
[0158] Tertiary-bromoester alcohol (TBEA in scheme 5) (1.8 g, 8.6
mmol), TEA (1.8 mL, 12.9 mmol) and DMAP (52.6 mg, 0.4 mmol) were
transferred to a 100 mL two-necked round-bottomed flask, equipped
with a magnetic stirrer bar, dropping funnel and septa cap
containing outlet. The flask was then deoxygenated using nitrogen.
Dry DCM (30 mL) was added via syringe under nitrogen. Xant e (5.0
g, 9.9 mmol) was deoxygenated using nitrogen inside the sealed
dropping funnel. Dry DCM (10 mL) was added via syringe. Xant e was
added dropwise. The flask was cooled in an ice bath during this
addition. The reaction was left stirring overnight. The resulting
brown solution was washed (1.times.80 mL distilled water and
2.times.80 mL brine). The organic layer was dried over MgSO.sub.4,
and the solid filtered off. The solvent was reduced and the product
was run through an automated flash column with a starting eluent of
100:0 hexane: ethyl acetate increasing to 20:80. Product fractions
collected and solvent removed. The product was further washed with
DCM to remove residual ethyl acetate, and solvent removed again.
The resulting yellow/brown oil was left in a high vacuum vessel
overnight to remove any residual solvent. Yield (40%). .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta.: 1.28 (s, 3H), 1.43 (t, 6H), 1.95 (s,
6H), 2.78 (t, 4H), 3.37 (t, 4H), 4.25 (m, 411), 4.42 (m, 4H), 4.65
(q, 4H). Mass spec: m/z=703.0 [M+Na].sup.+.
[0159] 1.4.6 G1, G2, G3 Xanthate Dendron Synthesis Using bisMPA
Backbone
[0160] For key references relating to the synthesis of bis-MPA
dendrimers, refer to the following: [0161] Macromolecules 2002, 35,
8307-8314 [0162] J. Am. Chem. Soc., 2001, 123, 5908-5917 [0163] J.
Am. Chem. Soc., 2009, 131, 2906-2916
[0164] For preparation of benzylidene protected bis-MPA anhydride
follow: [0165] J. Am. Chem. Soc. 2001, 123, 5908-5917
[0166] For preparation of DPTS 4-(Dimethylamino)pyridinium
4-toluenesulfonate follow: [0167] J. S. Moore, S. I. Stupp,
Macromolecules, 1990, 23, 65
[0168] For preparation of 2-hydroxyethyl 2-bromo-2-methylpropanoate
follow: [0169] J. Mater. Chem., 2011, 21, 18623-18629
Preparation of Xanthate Based Carboxylic Acid Building Block
##STR00018##
[0171] Synthesis of 2-((Ethoxycarbonothioyl)thio)acetic acid 1--A
500 mL round-bottomed flask equipped with a dropping funnel was
charged with a magnetic stirrer bar, potassium ethyl xanthogenate
(26.77 g, 167 mmol), and acetone (75 mL). A solution of
2-bromoacetic acid (19.31 g, 103 mmol) in acetone (40 mL) was added
dropwise at room temperature over a period of 60 min. Stirring was
continued overnight at room temperature. Solids were removed by
filtration to afford a clear pale yellow solution. The solids on
the funnel were washed with acetone (total of 50 mL). The combined
washing and filtrate solutions were concentrated under vacuum to
furnish a yellow viscous liquid that was dissolved in
dichloromethane (150 mL). This solution was washed twice with brine
(100 mL), and the organic phase was dried over MgSO4 and evaporated
to dryness to afford 18.75 g (75%) of a white solid. .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta.=1.43 (t, J=7.32 Hz, 3H), 3.98 (s,
2H) 4.67 (q, J=7.25 Hz, 2H), 4.53 .sup.13C NMR (100 MHz,
CDCl.sub.3): .delta.=13.68, 37.60, 70.93, 174.30, 212.01
##STR00019##
General Procedure for Dendon Growth (2, 4 and 6)--
[0172] To a 500 ml oven-dried round-bottom flask equipped with a
magnetic stirrer (under nitrogen atmosphere), the benzylidene
protected anhydride, the hydroxyl-terminated dendron (generation 0
through to 3), and 4-dimethylaminopyridine (DMAP) were all
dissolved in a 1:1 ratio of CH.sub.2Cl.sub.2:pyridine (v/v). After
stirring at room temperature for over 12 h, approximately 2 mL of
water was added and the reaction was stirred for an additional 18 h
in order to quench the excess anhydride. The product was isolated
by diluting the mixture with CH.sub.2Cl.sub.2 (150 mL) and washing
with 1 M NaHSO4 (3.times.150 mL), saturated aqueous NaHCO.sub.3
(2.times.150 mL), and brine (150 mL). The organic layer was dried
over MgSO4 and evaporated to dryness. Any residual solvent was
removed under high vacuum overnight to yield a white foam with a
typical yield greater than 95%.
General Procedure for Deprotection of Benzylidene by Hydrogenation
(3, 5 and 7)--
[0173] To a reactor suitable for medium pressure hydrogenation
fitted with a magnetic stirrer, the benzylidene protected dendrimer
was dissolved in a 1:1 mixture of CH.sub.2Cl.sub.2: MeOH (v/v).
Pd(OH).sub.2 on carbon (20%) was added and the reactor was
evacuated and back-filled with hydrogen three times (H.sub.2
pressure: 10 bar). After vigorous stirring for 16 h, the reaction
mixture was filtered through celite using a Buchner funnel and the
filtrate was evaporated to dryness on a rotary evaporator under
vacuo. The product was isolated as white foam in quantitative
yields.
##STR00020## ##STR00021##
General Procedure for Surface Group Modification to Xanthates (8, 9
and 10)--
[0174] To a 500 mL oven-dried round-bottom flask equipped with a
magnetic stirrer (under nitrogen atmosphere), the
hydroxyl-terminated dendron (generation 0 through to 3),
2-((Ethoxycarbonothioyl)thio)acetic acid 1, and
4-(Dimethylamino)pyridinium 4-toluenesulfonate (DPTS) were all
dissolved in the minimum amount of CH.sub.2Cl.sub.2. After the
reaction flask was flushed with nitrogen, DCC was added. Stirring
at room temperature was continued for 18 h under a nitrogen
atmosphere. Once the reaction was complete the DCC-urea was
filtered off and washed with a small volume of CH.sub.2Cl.sub.2.
The crude product was purified by liquid chromatography on silica
gel, eluting with hexane gradually increasing to 40:60 ethyl
acetate/hexane to give a yellow viscous oil.
[0175] General Procedure for Deprotection of Para-Toluene Sulfonyl
Ester (TSe) by DBU (11, 12 and 13)--
[0176] To an oven-dried round-bottom flask equipped with a magnetic
stirrer, the benzylidene protected dendrimer was dissolved in 50 mL
of CH.sub.2Cl.sub.2. 1.4 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) was added. The reaction was stirred under a nitrogen
atmosphere for 3 hrs and monitored until completion by TLC (60:40
hexane:ethyl acetate). The product was isolated by diluting the
mixture with CH.sub.2Cl.sub.2 (100 mL) and washing with 1 M
NaHSO.sub.4 (2.times.100 mL). The organic layer was dried over
MgSO4 and evaporated to dryness. The product was then precipitated
three times from hexanes. Any residual solvent was removed under
high vacuum to yield a viscous oil with typical yields greater than
95%.
[0177] General Procedure for Focal Point Modification to an ATRP
Initiator by DCC/DPTS Couplings (14, 15 and 16)--
[0178] To a 500 mL oven-dried round-bottom flask equipped with a
magnetic stirrer (under nitrogen atmosphere), the carboxylic acid
focal point xanthate dendron (generation 0 through to 3),
2-hydroxyethyl 2-bromo-2-methylpropanoate, and
4-(Dimethylamino)pyridinium 4-toluenesulfonate (DPTS) were all
dissolved in the minimum amount of CH.sub.2Cl.sub.2. After the
reaction flask was flushed with nitrogen, DCC was added. Stirring
at room temperature was continued for 18 h under a nitrogen
atmosphere. Once the reaction was complete the DCC-urea was
filtered off and washed with a small volume of CH.sub.2Cl.sub.2.
The crude product was purified by liquid chromatography on silica
gel, eluting with hexane gradually increasing to 40:60 ethyl
acetate/hexane to give a dark yellow viscous oil.
[0179] Synthesis of 2--The dendron growth step was carried out as
described above, using para-toluene sulfonyl ethanol (10 g, 50
mmol), benzylidene anhydride (42.65 g, 100 mmol, 2 equiv) and DMAP
(2.57 g, 21 mmol)) dissolved in 220 mL of dry CH.sub.2Cl.sub.2 and
120 mL of pyridine, and stirred for 16 h at room temperature.
Yield: 19.78 g, white foam (98%). .sup.1H NMR (400 MHz,
CDCl.sub.3): 8=0.96 (s, 3H), 2.43 (s, 3H), 3.47 (t, J=6.3 Hz, 2H),
3.60 (d, J=11.6 Hz, 2H) 4.47 (t, J=6.26 Hz, 2H), 4.53 (d, J=11.54
Hz, 2H), 5.43 (s, 1H), 7.33 (m, 5H), 7.41 (m, 2H), 7.81 (d, J=8.42
Hz, 2H). .sup.13C NMR (100 MHz, CDCl.sub.3): 5=17.51, 21.64, 42.46,
55.13, 58.20, 73.32, 101.72, 126.15, 128.19, 128.23, 129.01,
130.09, 136.01, 145.11, 149.86, 173.52.
[0180] Synthesis of 3--Deprotection of 2 (5.58 g, 13.60 mmol) in
210 mL of CH.sub.2Cl.sub.2: MeOH (1:1, v/v) was carried out as
above for 16 h at room temperature under 10 bar H.sub.2 atmosphere.
0.55 g Pd(OH).sub.2 was used. Yield: 4.3 g, white foam (99%).
.sup.1H NMR (400 MHz, CD.sub.3OD): .delta.=1.03 (s, 3H), 2.45 (s,
3H), 3.50 (dd, J=42.53, 10.95 Hz, 4H), 3.59 (t, J=5.98 Hz, 211),
4.39 (t, J=5.85 Hz, 2H), 7.47 (d, 211), 7.82 (d, 2H). .sup.13C NMR
(100 MHz, CD.sub.3OD): .delta.=17.07, 21.61, 51.58, 55.90, 58.93,
65.66, 129.30, 131.22, 137.76, 146.71, 175.89.
[0181] Synthesis of 4--The dendron growth step was carried out as
described above, using 3 (4.10 g, 12.96 mmol), benzylidene
anhydride (16.58 g, 39 mmol, 3 equiv) and DMAP (0.71 g, 5.38 mmol))
all dissolved in 70 mL of dry CH.sub.2Cl.sub.2 and 35 mL of
pyridine, and stirred for 16 h at room temperature. Yield: 8.68 g,
white foam (94%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=0.95
(s, 6H), 1.09 (s, 3H), 2.37 (s, 3H), 3.10 (t, J=5.8 Hz, 2H), 3.60
(d, J=12.45 Hz, 4H) 4.20 (m, 6H), 4.56 (t, J=9 Hz, 4H), 5.42 (s,
2H), 7.30 (m, 8H), 7.39 (m, 4H), 7.68 (d, J=8.43 Hz, 2H). .sup.13C
NMR (100 MHz, CDCl.sub.3): 6=17.33, 17.72, 21.56, 42.60, 46.70,
54.65, 58.32, 65.20, 73.46, 73.53, 101.63, 126.12, 128.05, 128.16,
128.91, 130.00, 136.29, 137.78, 145.00, 172.00, 173.17. Accurate MS
Calc'd for C.sub.38H O.sub.12S [M+Na].sup.+=747.2451. Found:
[M+Na].sup.+=742.2426, ES MS: [M+Na].sup.+=747.20,
[M+K].sup.+=763.2.
[0182] Synthesis of 5--Deprotection of 4 (7.90 g, 10.90 mmol) in
190 mL of CH.sub.2Cl.sub.2: MeOH (1:1, v/v) was carried out as
above for 16 h at room temperature under 10 bar H.sub.2 atmosphere.
0.40 g Pd(OH).sub.2 was used. Yield: 5.93 g, white foam (99%).
.sup.1H NMR (400 MHz, CD.sub.3OD): .delta.=1.15 (s, 9H), 2.48 (s,
3H), 3.57-3.69 (m, 10H), 4.11 (dd, J=31.18, 9.37 Hz) 4H), 4.46 (t,
J=5.77 Hz, 21H), 7.49 (d, J=8.81 Hz, 2H), 7.85 (d, J=8.39 Hz, 2H).
.sup.13C NMR (100 MHz, CD.sub.3OD): .delta.=15.38, 15.94, 19.72,
45.76, 49.91, 53.92, 57.75, 63.95, 64.25, 127.40, 129.41, 136.02,
144.82, 171.81, 173.94. Accurate MS Calc'd for
C.sub.24H.sub.36O.sub.12S [M+Na].sup.+ m/z=571.1825, [M+Na].sup.+
m/z=571.1821. Found ES MS: [M+Na].sup.+=571.2,
[M+K].sup.+=587.2.
[0183] Synthesis of 6--The dendron growth step was carried out as
described above, using 5 (2.58 g, 4.56 mmol), benzylidene anhydride
(11.67 g, 27.36 mmol, 6 equiv) and DMAP (0.35 g, 2.83 mmol)) all
dissolved in 46 mL of dry CH.sub.2Cl.sub.2 and 23 mL of pyridine,
and stirred for 16 h at room temperature. Yield: 6.23 g, white foam
(94%). .sup.1H NMR (400 MHz, CDCl.sub.3): S=0.93 (m, 15H), 1.19 (s,
6H), 2.39 (s, 3H), 3.28 (t, J=6.38 Hz, 2H), 3.58 (d, J=11.82 Hz,
8H), 3.94 (dd, J=30.95, 11.33 Hz, 4H), 4.33 (m, 10H), 4.56 (d, J=12
Hz, 8H), 5.40 (s, 4H), 7.30 (m, 14H), 7.39 (m, 8H), 7.74 (d, J=8.52
Hz, 2H). .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.=16.85, 17.66,
21.59, 42.59, 46.30, 46.87, 54.58, 58.22, 65.14, 65.70, 73.44,
73.52, 101.68, 126.20, 128.07, 128.13, 128.88, 130.04, 136.26,
137.82, 144.50, 171.63, 171.83, 173.20. ES MS: [M+Na]+=1387.5,
[M+K].sup.+=1403.5 Synthesis of 7--Deprotection of 6 (5.80 g, 4.25
mmol) in 200 mL of CH.sub.2Cl.sub.2: MeOH (1:1, v/v) was carried
out as above for 16 h at room temperature under 10 bar Hz
atmosphere. 0.29 g Pd(OH).sub.2 was used. Yield: 4.31 g, white foam
(99%). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.=1.15 (m, 15H),
1.28 (s, 6H), 2.48 (s, 3H), 3.62 (m, 18H), 4.24 (m, 12H), 4.48 (t,
J=6.14 Hz, 2H), 7.49 (d, J=8.10 Hz, 2H), 7.85 (d, J=8.19 Hz, 2H).
ES MS: [M+Na].sup.+=1035.4, [M+K].sup.+=1051.4
[0184] Synthesis of 8--1, 4.65 g (25.80 mmol), and 2.72 g (8.60
mmol) of 3, 1.01 g (3.44 mmol) of DPTS, and 5.86 g (28.38 mmol) of
DCC were allowed to react according to the general esterification
procedure in 40 mL of dry CH.sub.2Cl.sub.2 for 18 h. The crude
product was purified by liquid chromatography on silica gel,
eluting with hexane gradually increasing to 40:60 ethyl
acetate/hexane to give 6 as a yellow viscous oil 4.6 g (84%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=1.16 (s, 3H), 1.42 (t,
J=7.15, 6H), 2.46 (s, 3H), 3.44 (t, J=6.3 Hz, 2H), 3.91 (s, 4H),
4.18 (dd, J=31.72, 11.36 Hz, 4H) 4.46 (t, J=6.03 Hz, 2H), 4.64 (q,
J=7.12 Hz, 4H), 7.39 (d, J=8.23, 2H), 7.80 (d, J=7.70, 2H).
.sup.13C NMR (100 MHz, CDCl.sub.3): .delta.=13.74, 17.56, 21.67,
37.70, 54.97, 58.36, 60.39, 66.21, 70.91, 128.12, 130.18, 136.18,
145.28, 167.33, 171.80, 212.57.
[0185] ES MS: [M+Na].sup.4=663.0, [M+K].sup.+=679.0
[0186] Synthesis of 9--1, 9.97 g (55.32 mmol), and 5.06 g (9.22
mmol) of 5, 2.17 g (7.38 mmol) of DPTS, and 12.56 g (60.85 mmol) of
DCC were allowed to react according to the general esterification
procedure in 170 mL of dry CH.sub.2Cl.sub.2 for 18 h. The crude
product was purified by liquid chromatography on silica gel,
eluting with hexane gradually increasing to 50:50 ethyl
acetate/hexane to give 6 as a orange viscous oil 9.65 g (88%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=1.20 (s, 3H), 1.25 (s,
6H), 1.42 (t, J=7.16, 12H), 2.47 (s, 3H), 3.44 (t, J=5.97 Hz, 2H),
3.94 (s, 8H), 4.25 (m, 12H) 4.46 (t, J=5.90 Hz, 2H), 4.64 (q,
J=7.01 Hz, 8H), 7.40 (d, J=8.51, 211), 7.82 (d, J=8.31, 2H).
[0187] Synthesis of 10--See the general procedure
[0188] Synthesis of 11--The removal of the para-toluene sulfonyl
protecting group was carried out as described above, using 8 (4.60
g, 7.18 mmol, 1.0 equiv), and DBU (1.40 mL, 9.33 mmol, 1.3 equiv)
dissolved in 80 mL of CH.sub.2Cl.sub.2 and stirred for 3 h. The
reaction was monitored using TLC, 40:60 ethyl acetate/hexane.
Yield: 3.29 g, orange viscous oil (99%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.=1.32 (s, 3H), 1.42 (t, J=7.05, 6H), 2.47 (s,
3H), 3.94 (s, 4H), 4.33 (dd, J=: 39.96, 11.14 Hz, 2H), 4.64 (q,
J=7.14 Hz, 4H). .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.=13.74,
17.86, 37.74, 46.06, 66.13, 70.87, 167.45, 177.80, 212.53. ES MS:
[M+Na].sup.+:=481.0
[0189] For the synthesis of 12 and 13,--see the general
procedure
[0190] 1.4.7 G1 Morpholine Dendron Initiator (G1 ML Br)
##STR00022##
[0191] 1,1'-Carbonyldiimidazole (6.0994 g, 37.62 mmol) was added to
a 2-neck round bottom flask, which was equipped with a magnetic
stirring, condenser, and a N.sub.2 inlet. Anhydrous toluene (60 ml)
and N-(2-Hydroxypropyl)morpholine, 1, (5.35 ml, 37.62 mmol) were
added and the reaction was heated to 60.degree. C. The AB.sub.2
brancher (3.5603 g, 18.81 mmol) dissolved in anhydrous toluene (6.0
ml) was added after 3 hours of reaction. After a further 16 hours
the reaction was stopped, the toluene removed in vacuo, the crude
mixture was dissolved in dichloromethane (100 ml) and washed with
NaOH solution (pH 14) (3.times.100 ml). The organic phase was dried
over Na.sub.2SO.sub.4 the solvent removed in vacuo and the
resulting yellow oil was dried further under high vacuum to give 2,
(75%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 1.13 (d, 3H),
1.22 (d, 6H), 1.67 (m, 4H), 2.25-2.65 (br m, 18H), 3.22 (m, 4H),
3.68 (m, 8H), 3.79 (m, 1H), 4.98 (m, 2H), 5.29 and 5.40 (br s, NH).
.sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 19.30, 20.83, 27.58,
27.76, 39.59, 52.28, 54.39, 62.86, 64.08, 67.36, 67.96, 68.12,
156.73. Calcd.: [M)]m/z=531.36. Found: ES-MS: [M+H].sup.+=532.4,
[M+Na].sup.+=554.4. Found, C, 56.58; H, 9.24; N, 13.23%.
C.sub.25H.sub.49N.sub.5O.sub.7 requires, C, 56.47; H, 9.29; N,
13.17%.
[0192] 2, (7.546 g, 14.2 mmol) was dissolved in DCM (150 ml) and
bubbled with N.sub.2 for 20 minutes. 4-(Dimethylamino)pyridine
(DMAP) (86.7 mg, 0.7 mmol) and triethylamine (TEA) (2.37 ml, 17.0
mmol) were added and the reaction vessel was cooled to 0.degree. C.
.alpha.-Bromoisobutyryl bromide (1.93 ml, 15.6 mmol) was added
dropwise, then the reaction was warmed to room temperature for 16
hours. The reaction colour changed from pale yellow to a dark peach
colour over this time period. The organic phase was washed with a
saturated solution of NaHCO.sub.3 (3.times.150 ml) and distilled
water (3.times.150 ml), dried over Na.sub.2SO.sub.4 and the solvent
removed in vacuo to give a crude brown coloured oil. This was
purified by silica column chromatography with a mobile phase of
EtOAc:MeOH (4:1), (Rf=0.49) to give a light brown coloured oil, 3,
(49%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 1.24 (m, 9H),
1.65 (m, 4H), 1.92 (d, 6H), 2.26-2.70 (br m, 18H), 3.20 (m, 4H),
3.69 (m, 8H), 4.98 (m, 2H), 5.06 (m, 1H) 5.36 (br s, NH). .sup.13C
NMR (100 MHz, CDCl.sub.3): S. Calcd.: [M].sup.+ m/z=679.32. Found:
ES-MS: [M+H].sup.+=680.3, [M+Na].sup.+=702.3. Found, C, 50.87; H,
7.95; N, 10.37%. C.sub.29H.sub.54N.sub.5O.sub.8Br requires, C,
51.17; H, 8.00; N, 10.29%.
[0193] 1.4.8 G1 bisMPA Dendron Initiator (G1 MPA Br)
##STR00023##
[0194] 1,1'-Carbonyldiimidazole (9.729 g, 60.0 mmol) was weighed
into a 3-neck round bottom flask fitted with a N.sub.2 inlet,
magnetic stirrer and condenser. Anhydrous THF (120 ml) was added
via double ended needle. The reaction was heated to 60.degree. C.
and iPbisMPA (10.4514 g, 60.0 mmol) was added under a positive
N.sub.2 flow. Reaction could be observed by the evolution of
CO.sub.2 and the reaction became effervescent. To avoid too much
effervescence the iPbisMPA was added slowly, approx. 2 g at a time
once the effervescence had died down. After 3 hours the reaction
mixture was bubbled through with N.sub.2 to ensure any residual
CO.sub.2 had been removed from the reaction medium and flask. The
AB.sub.2 brancher (5.949 g, 30.0 mmol) was added dropwise in
anhydrous THF (20 ml), after a further 18 hours the reaction was
stopped and THF removed in vacuo. The crude residue was dissolved
in DCM (125 ml) and washed with NaOH solution (pH14) (3.times.125
ml) and distilled water (125 ml). The organic phase was dried over
Na.sub.2SO.sub.4 and the DCM was removed in vacuo then under high
vacuum, to give a pale yellow oil, 1, (78%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 1.02 (s, 6H), 1.10 (d, 3H), 1.42 (s, 6H), 1.47
(s, 6H), 1.70 (m, 4H), 2.32 (d of d of d, 2H), 2.45 (m, 2H), 2.63
(m, 2H), 3.34 (q, 4H), 3.75 (m, 5H), 3.92 (d, 4H). .sup.13C NMR
(100 MHz, CDCl.sub.3): .delta. 18.30, 19.11, 20.38, 27.57, 29.08,
37.87, 40.59, 51.85, 63.00, 63.64, 67.54, 98.93, 175.24. Calcd.:
[M].sup.+ m/z=501.34. Found: CI-MS: [M+H]+=502.7. Found, C, 59.86;
H, 9.41; N, 8.18%. C.sub.25H.sub.47N.sub.3O.sub.7 requires, C,
59.86; H, 9.44; N, 8.38%.
[0195] G1 MPA OH dendron (5.127 g, 10.2 mmol) was weighed into a
round bottom flask and dissolved in DCM (70 ml) and degassed with
dry nitrogen for 10 min. DMAP (62 mg, 0.51 mmol) and TEA (1.71 ml,
12.3 mmol) were added, the vessel was maintained under a positive
nitrogen flow and cooled to 0.degree. C. .alpha.-Bromoisobutyryl
bromide (1.38 ml, 11.2 mmol) was added dropwise then was warmed to
room temperature for 18 hours. The reaction was a light yellow
colour to begin with and changed to a slightly darker yellow over
time, no precipitate was observed. The reaction mixture was washed
with a saturated NaHCO.sub.3 solution (3.times.100 ml) and water
(3.times.100 ml), dried over Na.sub.2SO.sub.4 and concentrated in
vacuo to give G MPA Br, 2, (54%) as a yellow viscous oil. .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 1.04 (s, 6H), 1.24 (d, 3H), 1.42
(s, 6H), 1.46 (s, 6H), 1.67 (m, 4H), 1.91 (s, 6H), 2.40-2.67 (m,
6H), 3.31 (m, 4H), 3.74 (d, 4H), 3.96 (d, 4H), 5.05 (m, 1H).
.sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 18.35, 18.43, 27.58,
28.48, 31.16, 37.85, 40.69, 52.09, 56.54, 59.54, 67.44, 67.51,
70.94, 98.74, 171.67, 175.12. Calcd.: [M].sup.+ m/z=649.29. Found:
ES-MS: [M+H]:=650.3. Found, C, 53.61; H, 8.16; N, 6.42%.
C.sub.29H.sub.53BrN.sub.3O.sub.7 requires, C, 53.53; H, 8.06; N,
6.46%.
[0196] 1.4.9 G1-A Tertiary Amine Dendron Initiator
Synthesis of G1-A Dendron
##STR00024##
[0198] 1-dimethylamino-2-propanol (2.4758 g, 24 mmol, 4 eq.) was
added to a 100 mL 2 necked round-bottomed flask containing
anhydrous toluene (20 mL) and fitted with a reflux condenser,
magnetic stirrer and a positive flow of N.sub.2. The solution was
stirred at room temperature and CDl (1.9458 g, 12 mmol, 2 eq.) was
added. The mixture was heated to 60.degree. C. with stirring for 6
hours. AB.sub.2 brancher (1.1358 g, 6 mmol, 1 eq.) dissolved in
anhydrous toluene (5 mL) was deoxygenated using a N.sub.2 purge for
10 minutes and was added drop wise while the solution was stirred
and the temperature was maintained at 60.degree. C. The reaction
was stirred for a further 18 hours at 60.degree. C., and then
allowed to cool to room temperature. The solution was concentrated
in vacuo, and the remaining oil was dissolved in DCM (30 mL) and
washed with 1M NaOH solution (3.times.30 mL). The solution was
dried with anhydrous Na.sub.2SO, filtered and concentrated in vacuo
to give G1-A as a viscous liquid. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.25 (m, 9H), 1.64 (m, 3H), 2.05-2.67 (m, 22H), 3.20 (m,
3H), 3.78 (m, 1H), 4.89 (m, 2H). m/z (ES MS) 448.4 [M+H]+, 470.3
[M+Na]+.
Synthesis of G1-A Dendron Initiator
##STR00025##
[0200] G1-A (0.8944 g, 2 mmol, 1 eq.), TEA (0.2833 g, 2.8 mmol, 1.4
eq.) and DMAP (24.43 mg, 0.2 mmol, 0.1 eq.) were added to a 100 mL
2 necked round-bottomed flask containing DCM (40 mL). The flask was
deoxygenated under a positive N.sub.2 purge for 10 minutes.
.alpha.-bromoisobutyryl bromide (0.4828, 0.26 mL, 2.7 mmol, 1.05
eq.) was added drop wise while the solution was stirring in an ice
bath under a positive flow of N.sub.2. The reaction mixture was
allowed to warm to room temperature and left stirring overnight.
The organic phase was washed with saturated sodium hydrogen
carbonate (NaHCO.sub.3) solution (3.times.30 mL). The solution was
dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in
vacuo to give initiator G1-A as a viscous yellow liquid. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 1.24 (m, 9H), 1.64 (m, 4H), 1.92
(d of d, 8H), 2.05-2.05-2.67 (m, 22H), 3.21 (m, 4H), 4.89 (m, 2H),
5.06 (m, 1H). m/z (ES MS) 596.3 [M+H]+, 617.3 [M+Na]+, 639.2
[M+K]+.
[0201] 1.4.10 G1-D Tertiary Amine Dendron Initiator
Synthesis of G1-D Dendron (HR2-136)
##STR00026##
[0203] 2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was
added to a 50 mL round 2 necked round-bottomed flask containing IPA
(12 mL). The flask was deoxygenated under a positive N.sub.2 purge
for 10 minutes. 1-amino-2-propanol (0.5246 g, 7.0 mmol, 1 eq.)
dissolved in IPA (12 mL) was added drop wise while the solution was
stirring in an ice bath under a positive flow of N.sub.2. The final
mixture was stirred for a further 10 minutes at 0.degree. C. before
being allowed to warm to room temperature and left stirring for 48
hrs. The solvent was removed and the product left to dry in vacuo
overnight. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.08 (d, 3H),
2.18-2.62 (m, 22H), 2.69 (m, 2H), 2.89 (m, 2H), 3.77 (m, 1H), 4.16
(m, 4H). m/z (ES MS) 362.3 [M+H]+, 384.3 [M+Na]+.
Synthesis of G1-D Dendron Initiator (HR2-143)
##STR00027##
[0205] G1-D dendron (1.1207 g, 10.86 mmol, 1 eq.), TEA (1.5390 g,
15.2 mmol, 1.4 eq.) and DMAP (132.7 mg, 1.086 mmol, 0.1 eq.) were
added to a 250 mL 2 necked round-bottomed flask containing DCM (160
mL). The flask was deoxygenated under a positive N.sub.2 purge for
10 minutes. .alpha.-bromoisobutyryl bromide (2.622 g, 1.4 mL, 11.4
mmol, 1.05 eq.) was added drop wise while the solution was stirring
in an ice bath under a positive flow of N.sub.2. The reaction
mixture was allowed to warm to room temperature and left stirring
overnight. The organic phase was washed with saturated sodium
hydrogen carbonate (NaHCO.sub.3) solution (3.times.160 mL). The
solution was dried with anhydrous Na.sub.2SO.sub.4 and the product
left to dry in vacuo overnight. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.22 (d, 3H), 1.89 (m, 6H), 2.24-2.69 (m, 22H), 2.83 (m,
4H), 4.20 (m, 4H), 5.0 (m, 2H). m/z (ES MS) 510.2 [M+H]+, 534.2
[M+Na]+.
Synthesis of G2-D Dendron (HR2-116)
##STR00028##
[0207] 2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was
added to a 50 mL round 2 necked round-bottomed flask containing IPA
(12 mL). The flask was 0.14 deoxygenated under a positive N.sub.2
purge for 10 minutes. Bis(3-aminopropyl)amino)propan-2-ol (1.3221
g, 6.984 mmol, 1 eq.) dissolved in IPA (12 mL) was added drop wise
while the solution was stirring in an ice bath under a positive
flow of N.sub.2. The final mixture was stirred for a further 10
minutes at 0.degree. C., allowed to warm to room temperature and
left stirring for 48 hrs. The solvent was removed and the product
left to dry in vacuo overnight. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.13 (d, 3H), 1.67 (m, 4H), 2.26-2.65 (m, 50H), 2.77 (m,
8H), 3.87 (m, 1H), 4.17 (m, 8H). m/z (ES MS) 762.6 [M+H]+, 784.6
[M+Na]+.
[0208] 1.4.11 G2-D Tertiary Amine Dendron Initiator
Synthesis of G2-D Dendron Initiator (HR2-121)
##STR00029##
[0210] G2-dendron (5.1431 g; 6.749 mmol, 1 eq.), TEA (0.9561 g,
9.449 mmol, 1.4 eq.) and DMAP (82.5 mg, 0.6749 mmol, 0.1 eq.) were
added to a 250 mL 2 necked round-bottomed flask containing DCM (160
mL). The flask was deoxygenated under a positive N.sub.2 purge for
10 minutes. .alpha.-bromoisobutyryl bromide (1.629 g, 0.88 mL,
7.087 mmol, 1.05 eq.) was added drop wise while the solution was
stirring in an ice bath under a positive flow of N.sub.2. The
reaction mixture was allowed to warm to room temperature and left
stirring overnight. The organic phase was washed with saturated
sodium hydrogen carbonate (NaHCO.sub.3) solution (3.times.160 mL).
The solution was dried with anhydrous Na.sub.2SO.sub.4 and the
product left to dry in vacuo overnight. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 1.26 (d, 3H), 1.56 (m, 411), 1.91 (m, 6H),
2.22-2.67 (m, 50H), 2.76 (m, 8H), 4:19 (m, 8H), 5.04 (m, 1H). m/z
(ES MS) 912.5 [M+H]+, 934.5 [M+Na]+, 950.5 [M+K]+.
2. Polydendrons
100% Dendron Initiated Branched Polymers
[0211] 2.1 HPMA (Hydrophobic Polymer Core)
[0212] 2.1.1 Hydrophobic Dendron Initiators
[0213] 2.1.1.1 Aromatic Dendrons G1 and G2 DBOP Br
[0214] In a typical experiment, G1 DBOP Br (0.291 g, 0.69 mmol) o
G2 DBOP Br (0.648 g, 0.69 mmol) and HPMA (targeted DP=50) (5.0 g,
34.7 mmol) were weighed into a round bottom flask. EGDMA (105
.mu.l, 0.55 mmol) was added and the flask was equipped with
magnetic stirrer bar, sealed and degassed by bubbling with N.sub.2
for 20 minutes and maintained under N.sub.2 at 30.degree. C.
Anhydrous methanol was degassed separately and subsequently added
to the monomer/initiator/brancher mixture via syringe to give a 50%
v/v mixture with respect to the monomer. The catalytic system;
Cu(I)Cl (0.069 g, 0.69 mmol) and 2,2'-bipyridyl (bpy) (0.217 g,
1.39 mmol), were added under a positive nitrogen flow in order to
initiate the reaction. The polymerisations were stopped when
conversions had reached over 98%. The polymerisations were stopped
by diluting with a large excess of tetrahydrofuran (THF), which
caused a colour change from dark brown to a bright green colour.
The catalytic system was removed using Dowex.RTM. Marathon.TM. MSC
(hydrogen form) ion exchange resin beads and basic alumina. The
resulting polymer was isolated by precipitation from the minimum
amount of THF into cold hexane. The [initiator]:[CuCl]:[bpy] molar
ratios in all polymerizations were 1:1:2. Other DPs targeted were
DP20 and DP100 with both G1 and G2 DBOP initiators.
[0215] 2.1.1.2 .sup.tBOC Dendrons G1 .sup.tBOC Br
[0216] The G1 .sup.tBOC Dendron initiator (100 mg, 0.186 mmol) was
added to a 25 mL round bottom flask equipped with a magnetic
stirrer bar, followed by the addition of 2,2-bipyridyl (58.1 mg,
0.372 mmol), EGDMA (35.1 mg, 0.177 mmol) and HPMA (1.34 g, 9.28
mmol). The reaction mixture was bubbled with N.sub.2 for 15
minutes. Degassed anhydrous methanol (3.45 mL) was added to the
flask, and its contents stirred and bubbled with N.sub.2 for a
further 5 minutes. Copper (I) chloride (18.4 mg, 0.186 mmol) was
quickly weighed out and added to the flask, instantly forming a
brown coloured mixture, which was stirred and bubbled with N.sub.2
for a further 5 minutes. A N.sub.2 pressure was then built up
within the flask, then N.sub.2 inlet removed, and the flask stirred
for 24 hours at 40.degree. C. Once the polymerisation was complete,
THF was added to the reaction flask to poison the Cu (I) catalyst,
forming a green coloured solution. The solution was passed through
an alumina (neutral) column to remove the catalytic system,
concentrated in vacuo, and precipitated into hexane. The
supernatant was decanted off, and the remaining white solid dried
overnight in a vac-oven.
[0217] 2.1.1.3 Xanthate Dendron Xant-G1
[0218] Xant-G1 initiator (578.0 mg, 0.868 mmol), BIPY (272.2 mg,
1.743 mmol), HPMA (6.3 g, 43.6 mmol,) and EGDMA (146.8 mg, 0.741
mmol) was transferred to 25 mL round-bottomed flask equipped with
stirrer bar and septa cap. The flask was deoxygenated using
nitrogen. Separately deoxygenated MeOH (12.9 mL, 38% w/v based on
HPMA) added via syringe. Once all reactants had dissolved, nitrogen
was bubbled through solution for 5 mins. Cu (I) Cl (86.3 mg, 0.868
mmol,) quickly measured out and added to round-bottomed flask.
Reaction mixture went from clear solution to deep red/brown.
Nitrogen was bubbled through solution for an additional 10 mins.
The reaction was then left to stir overnight under nitrogen.
Reaction mixture forms a deep red/brown viscous liquid on
completion. THF (20 mL) added to kill reaction. Once solution
turned a bright green colour, solution passed through a short
alumina column to remove copper catalyst, yielding a translucent
pale green solution. Solvent removed and resulting oily liquid
precipitated into cold hexane (approx. 50 mL, cooled in dry ice
bath). The resulting pale green crystals were filtered off and
washed with cold hexane. The sample was placed in a vacuum oven to
remove any residual solvent.
[0219] 2.1.2 Hydrophilic Dendrons
[0220] 2.1.2.1 G1-A Tertiary Amine Initiator
[0221] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (poly(HPMA).sub.50;
n.sub.DEAEMA/n.sub.Initiator: 50), bpy (173.3 mg, 1.1096 mmol, 2
eq.), HPMA (4 g, 27.7 mmol, 50 eq.), EGDMA (77.0 mg, 0.3883 mmol,
0.7 eq) and isopropanol (IPA) (38.9% v/v based on HPMA) were placed
into a 25 mL round-bottomed flask. The solution was stirred and
deoxygenated using a nitrogen (N.sub.2) purge for 15 minutes.
Cu(.sub.I)Cl (54.9 mg, 0.5548 mmol, 1 eq.) was added to the flask
and left to purge for a further 5 minutes. G1-A dendron initiator
(0.33 10 g, 0.5548 mmol, 1 eq.) was added to the flask under a
positive flow of N.sub.2, and the solution was left to polymerise
at 40.degree. C. Reactions were terminated when >99% conversion
was reached, as judged by .sup.1H NMR, by exposure to oxygen and
addition of THF. The catalyst residues were removed by passing the
mixture over a basic alumina column. THF was removed under vacuum
to concentrate the sample before precipitation into hexane and
drying in the vacuum oven overnight.
[0222] 2.1.2.2 G1 Morpholine Initiator (G1 ML Br)
[0223] G1 ML Br (0.378 g, 0.55 mmol) and HPMA (4.0 g, 27.7 mmol)
were weighed into a round bottom flask. EGDMA (73.2 .mu.l, 0.39
mmol) was added and the flask was equipped with magnetic stirrer
bar, sealed and degassed by bubbling with N.sub.2 for 20 minutes
and maintained under N.sub.2 at 30.degree. C. Isopropanol was
degassed separately and subsequently added to the
monomer/initiator/brancher mixture via syringe to give a 50 wt/wt %
mixture with respect to the monomer. The catalytic system; Cu(I)Cl
(0.055 g, 0.55 mmol) and 2,2'-bipyridyl (bpy) (0.173 g, 1.1 mmol),
were added under a positive nitrogen flow in order to initiate the
reaction. The polymerisations were stopped when conversions had
reached over 98%. The polymerisations were stopped by diluting with
a large excess of tetrahydrofuran (THF), which caused a colour
change from dark brown to a bright green colour. The catalytic
system was removed using Dowex.RTM. Marathon.TM. MSC (hydrogen
form) ion exchange resin beads and basic alumina. The resulting
polymer was isolated by precipitation from the minimum amount of
THF into cold hexane. The [initiator]:[CuCl]:[bpy] molar ratios in
all polymerizations were 1:1:2
[0224] 2.1.2.3 G1 bisMPA Initiator (G1 MPA Br)
[0225] G1 MPA Br (0.451 g, 0.69 mmol) and HPMA (5.0 g, 34.7 mmol)
were weighed into a round bottom flask. EGDMA (105 .mu.l, 0.55
mmol) was added and the flask was equipped with magnetic stirrer
bar, sealed and degassed by bubbling with N.sub.2 for 20 minutes
and maintained under N.sub.2 at 30.degree. C. Isopropanol was
degassed separately and subsequently added to the
monomer/initiator/brancher mixture via syringe to give a 50 wt/wt %
mixture with respect to the monomer. The catalytic system; Cu(I)Cl
(0.0687 g, 0.69 mmol) and 2,2'-bipyridyl (bpy) (0.217 g, 1.39
mmol), were added under a positive nitrogen flow in order to
initiate the reaction. The polymerisations were stopped when
conversions had reached over 98%. The polymerisations were stopped
by diluting with a large excess of tetrahydrofuran (THF), which
caused a colour change from dark brown to a bright green colour.
The catalytic system was removed using Dowex.RTM. Marathon.TM. MSC
(hydrogen form) ion exchange resin beads and basic alumina. The
resulting polymer was isolated by precipitation from the minimum
amount of THF into cold hexane. The [initiator]:[CuCl]:[bpy] molar
ratios in all polymerizations were 1:1:2.
[0226] 2.2 tBuMA (Hydrophobic Core)
[0227] 2.2.1 G1-A Tertiary Amine Dendron Initiator
[0228] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (poly(tBuMA).sub.50;
n.sub.DEAEMA/n.sub.Initiator: 50), bpy (175.7 mg, 1.1252 mmol, 2
eq.), tBuMA (4 g, 28.13 mmol, 50 eq.), EGDMA (105.9 mg, 0.5345
mmol, 0.95 eq) and aqueous isopropanol (7.5% water by volume)
(33.3% v/v based on tBuMA) were placed into a 25 mL round-bottomed
flask. The solution was stirred and deoxygenated using a nitrogen
(N.sub.2) purge for 15 minutes. Cu(.sub.I)Cl (55.7 mg, 0.5626 mmol,
1 eq.) was added to the flask and left to purge for a further 5
minutes. G1-A dendron initiator (0.3356 g, 0.5626 mmol, 1 eq.) was
added to the flask under a positive flow of N.sub.2, and the
solution was left to polymerise at 40.degree. C. Reactions were
terminated when >99% conversion was reached, as judged by
.sup.1H NMR, by exposure to oxygen and addition of THF. The
catalyst residues were removed by passing the mixture over a basic
alumina column. THF was removed under vacuum to concentrate the
sample before precipitation into hexane and drying in the vacuum
oven overnight.
[0229] 2.3 DEAEMA (Hydrophobic Core at Neutral/High pH, Hydrophilic
at Low pH)
2.3.1 G1-A Tertiary Amine Dendron Initiator
[0230] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (poly(DEAEMA).sub.50;
n.sub.DEAEMA/n.sub.Initiator: 50), bpy (134.9 mg, 0.8637 mmol, 2
eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886
mmol, 0.9 eq) and IPA.sup.37 (38.9% v/v based on DEAEMA) were
placed into a 25 mL round-bottomed flask. The solution was stirred
and deoxygenated using a N.sub.2 purge for 15 minutes. Cu(.sub.I)Cl
(42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left to
purge for a further 5 minutes. G1-A dendron initiator (0.2576 g,
0.4318 mmol, I eq.) was added to the flask under a positive flow of
N.sub.2, and the solution was left to polymerise at 40.degree. C.
Reactions were terminated when >99% conversion was reached, as
judged by .sup.1H NMR, by exposure to oxygen and addition of
acetone. The catalyst residues were removed by passing the mixture
over a basic alumina column. Acetone was removed under vacuum to
concentrate the sample before precipitation into cold petroleum
ether (40.degree. C.-60.degree. C.). The polymerisation conditions
and procedure is identical to those described for linear polymers
above and drying in the vacuum oven overnight.
[0231] 2.3.2 G0-D Tertiary Amine Dendron Initiator
[0232] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (poly(DEAEMA).sub.50;
n.sub.DEAEMA/n.sub.Initiator: 50), bpy (134.9 mg, 0.8637 mmol, 2
eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886
mmol, 0.9 eq) and IPA.sup.37 (38.9% v/v based on DEAEMA) were
placed into a 25 mL round-bottomed flask. The solution was stirred
and deoxygenated using a N.sub.2 purge for 15 minutes. Cu(.sub.I)Cl
(42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left to
purge for a further 5 minutes. G0-D dendron initiator (0.1089 g,
0.4318 mmol, 1 eq.) was added to the flask under a positive flow of
N.sub.2, and the solution was left to polymerise at 40.degree. C.
Reactions were terminated when >99% conversion was reached, as
judged by .sup.1H NMR, by exposure to oxygen and addition of
acetone. The catalyst residues were removed by passing the mixture
over a basic alumina column. Acetone was removed under vacuum to
concentrate the sample before precipitation into cold petroleum
ether (40.degree. C.-60.degree. C.) and drying in the vacuum oven
overnight. The polymerisation conditions and procedure is identical
to those described for linear polymers above.
[0233] 2.3.3 G1-D Tertiary Amine Dendron Initiator
[0234] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (poly(DEAEMA).sub.50;
n.sub.DEAEMA/n.sub.Initiator: 50), bpy (134.9 mg, 0.8637 mmol, 2
eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886
mmol, 0.9 eq) and IPA.sup.37 (38.9% v/v based on DEAEMA) were
placed into a 25 mL round-bottomed flask. The solution was stirred
and deoxygenated using a N.sub.2 purge for 15 minutes. Cu(.sub.I)Cl
(42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left to
purge for a further 5 minutes. G1-D dendron initiator (0.2204 g,
0.4318 mmol, I eq.) was added to the flask under a positive flow of
N.sub.2, and the solution was left to polymerise at 40.degree. C.
Reactions were terminated when >99% conversion was reached, as
judged by .sup.1H NMR, by exposure to oxygen and addition of
acetone. The catalyst residues were removed by passing the mixture
over a basic alumina column. Acetone was removed under vacuum to
concentrate the sample before precipitation into cold petroleum
ether (40.degree. C.-60.degree. C.) and drying in the vacuum oven
overnight. The polymerisation conditions and procedure is identical
to those described for linear polymers above.
[0235] 2.3.4 G2-D Tertiary Amine Dendron Initiator
[0236] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (poly(DEAEMA).sub.50;
n.sub.DEAEMA/n.sub.Initiator: 50), bpy (134.9 mg, 0.8637 mmol, 2
eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886
mmol, 0.9 eq) and IPA.sup.37 (38.9% v/v based on DEAEMA) were
placed into a 25 mL round-bottomed flask. The solution was stirred
and deoxygenated using a N.sub.2 purge for 15 minutes. Cu(.sub.I)Cl
(42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left to
purge for a further 5 minutes. G2-D dendron initiator (0.3934 g,
0.4318 mmol, I eq.) was added to the flask under a positive flow of
N.sub.2, and the solution was left to polymerise at 40.degree. C.
Reactions were terminated when >99% conversion was reached, as
judged by .sup.1H NMR, by exposure to oxygen and addition of
acetone. The catalyst residues were removed by passing the mixture
over a basic alumina column. Acetone was removed under vacuum to
concentrate the sample before precipitation into cold petroleum
ether (40.degree. C.-60.degree. C.) and drying in the vacuum oven
overnight. The polymerisation conditions and procedure is identical
to those described for linear polymers above.
[0237] 2.4 OEGMA (Hydrophilic Core)
[0238] 2.4.1 G1-A Tertiary Amine Dendron Initiator
[0239] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (poly(OEGMA).sub.50;
n.sub.DEAEMA/n.sub.Initiator: 50), bpy (83.3 mg, 0.5333 mmol, 2
eq.), OEGMA (4 g, 13.3 mmol, 50 eq.), EGDMA (50.2 mg, 0.2533 mmol,
0.95 eq) and aqueous isopropanol (7.5% water by volume) (33.3% v/v
based on OEGMA) were placed into a 25 mL round-bottomed flask. The
solution was stirred and deoxygenated using a nitrogen (N.sub.2)
purge for 15 minutes. Cu(.sub.I)Cl (26.4 mg, 0.2667 mmol, 1 eq.)
was added to the flask and left to purge for a further 5 minutes.
G1-A dendron initiator (0.1591 g, 0.2667 mmol, 1 eq.) was added to
the flask under a positive flow of N.sub.2, and the solution was
left to polymerise at 40.degree. C. Reactions were terminated when
>99% conversion was reached, as judged by .sup.1H NMR, by
exposure to oxygen and addition of THF. The catalyst residues were
removed by passing the mixture over a basic alumina column. THF was
removed under vacuum to concentrate the sample before precipitation
into cold hexane and drying in the vacuum oven overnight.
[0240] 2.5 Copolymer Synthesis
[0241] 2.5.1 G2-D Tertiary Amine Initiator,
pDEAEMA.sub.50-b-ptBuMA.sub.65-st-EGDMA.sub.0.9
[0242] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (poly(DEAEMA).sub.50;
n.sub.DEAEMA/n.sub.Initiator: 50), bpy (134.9 mg, 0.8637 mmol, 2
eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.) and isopropanol (IPA) (37.7%
v/v based on DEAEMA) were placed into a 50 mL round-bottomed flask.
The solution was stirred and deoxygenated using a nitrogen
(N.sub.2) purge for 15 minutes. Cu(.sub.I)Cl (42.8 mg, 0.4318 mmol,
1 eq.) was added to the flask and left to purge for a further 5
minutes. G2-D dendron initiator (0.3934 g, 0.4318 mmol, 1 eq.) was
added to the flask under a positive flow of N.sub.2, and the
solution was left to polymerise at 40.degree. C. In another 25 mL
round-bottomed flask, bpy (134.9 mg, 0.8637 mmol), tBuMA (4.0 g,
28.1 mmol, 65 eq.), EGDMA (77.0 mg, 0.3886 mmol, 0.9 eq) and
aqueous isopropanol (23.8% v/v based on tBuMA) were added. The
solution was stirred and deoxygenated using a nitrogen (N.sub.2)
purge for 15 minutes. Cu(.sub.I)Cl (42.8 mg, 0.4318 mmol, 1 eq.)
was added to the flask and left to purge for a further 5 minutes.
After the conversion of DEAEMA reached around 85%, the mixture from
the second flask was added into the first flask rapidly using a
syringe and taking care not to admit any air into the vessel. A
sample was taken immediately after the addition of the tBuMA
monomer solution for .sup.1H NMR analysis. The block
copolymerization reaction was carried out at ambient temperature
and samples were taken periodically from the reaction mixture for
.sup.1H NMR analysis. Reactions were terminated when >99%
conversion was reached, as judged by .sup.1H NMR, by exposure to
oxygen and addition of acetone. The catalyst residues were removed
by passing the mixture over a basic alumina column. Acetone was
removed under vacuum to concentrate the sample before precipitation
into cold petroleum ether (40.degree. C.-60.degree. C.) and drying
in the vacuum oven overnight.
TABLE-US-00001 TABLE 1 100% Dendron initiated polydendrons
Initiator Polymer EGDMA Generation Functionality Core (mol %) Mn
(gmol.sup.-1) Mw (gmol.sup.-1) PDI G1 DBOP pHPMA20 0.8 52 800 545
000 10.32 G1 DBOP pHPMA50 0.8 47 200 1 169 000 24.74 G1 DBOP
pHPMA100 0.8 69 300 1 354 500 19.54 G2 DBOP pHPMA20 0.8 153 000 1
565 000 10.23 G2 DBOP pHPMA50 0.8 59978 739440 12.33 G2 DBOP
pHPMA100 0.8 164 200 2 227 500 13.58 G1 tBOC pHPMA50 0.95 12282
45539 3.71 G1 Xanthate pHPMA50 0.85 63800 1070000 15 G1 Morpholine
pHPMA50 0.7 76687 454746 5.93 G1 bisMPA pHPMA50 0.8 77745 436461
5.61 G1-A t-amine pHPMA50 0.7 661180 966552 1.50 G1-A t-amine
ptBuMA50 0.95 150264 284002 1.90 G1-A t-amine pDEAEMA50 0.9 201497
244622 1.20 G1-A t-amine pOEGMA50 0.95 97082 216813 2.20 G0-D
t-amine pDEAEMA50 0.9 G1-D t-amine pDEAEMA50 0.9 G2-D t-amine
pDEAEMA50 0.9 125652 302557 2.40 G2-D t-amine pDEAEMA50- 0.9 129737
374192 2.90 b- tBuMA65- st-EGDMA
3. Mixed Initiator Systems
[0243] 3.1 Mixed Dendrons
[0244] 3.1.1 G1 and G2 tBOC Initiated pHPMA Core
[0245] The G1 .sup.tBOC Dendron initiator (67.9 mg, 0.126 mmol) and
G2 .sup.tBOC Dendron initiator (63.1 mg, 0.054 mmol) was added to a
25 mL round bottom flask equipped with a magnetic stirrer bar,
followed by the addition of 2,2-bipyridyl (56.2 mg, 0.360 mmol),
EGDMA (28.5 mg, 0.144 mmol) and HPMA (1.3 g, 9.0 mmol). The
reaction mixture was bubbled with N.sub.2 for 15 minutes. Degassed
anhydrous methanol (3.3 mL) was added to the flask, and its
contents stirred and bubbled with N.sub.2 for a further 5 minutes.
Copper (I) chloride (17.8 mg, 0.180 mmol) was quickly weighed out
and added to the flask, instantly forming a brown coloured mixture,
which was stirred and bubbled with N.sub.2 for a further 5 minutes.
A N.sub.2 pressure was built up within the flask, then N.sub.2
inlet then removed, and the flask stirred for 24 hours at
40.degree. C. Once the polymerisation was complete, THF was added
to the reaction flask to poison the Cu (I) catalyst, forming a
green coloured solution. The solution was passed through an alumina
(neutral) column to remove the catalytic system, concentrated in
vacuo, and precipitated into hexane. The supernatant was decanted
off, and the remaining white solid dried overnight in a
vac-oven.
[0246] 3.2 Mixed Dendron with Non-Dendron Initiator
[0247] 3.2.1 G2 DBOP Br and 750 PEG Initiated pHPMA Core
[0248] In a typical reaction, G2 DBOP Br (0.259 g, 0.28 mmol) and
750 PEG initiator (0.250 g, 0.28 mmol) (for a targeted ratio of G2
dendron:750 PEG of 50:50 mol %) were weighed into a round bottom
flask, followed by HPMA (4.0 g, 27.7 mmol). EGDMA (84 .mu.l, 0.44
mmol) was added and the flask was equipped with magnetic stirrer
bar, sealed and degassed by bubbling with N.sub.2 for 20 minutes
and maintained under N.sub.2 at 30.degree. C. Anhydrous methanol
was degassed separately and subsequently added to the
monomer/initiator/brancher mixture via syringe to give a 50 wt/wt %
mixture with respect to the monomer. The catalytic system; Cu(I)Cl
(0.055 g, 0.55 mmol) and 2,2'-bipyridyl (bpy) (0.173 g, 1.1 mmol),
were added under a positive nitrogen flow in order to initiate the
reaction. The polymerisations were stopped when conversions had
reached over 98%. The polymerisations were stopped by diluting with
a large excess of tetrahydrofuran (THF), which caused a colour
change from dark brown to a bright green colour. The catalytic
system was removed using Dowex.RTM. Marathon.TM. MSC (hydrogen
form) ion exchange resin beads and basic alumina. The resulting
polymer was isolated by precipitation from the minimum amount of
THF into cold hexane. The [initiator]:[CuCl]:[bpy] molar ratios in
all polymerizations were 1:1:2.
[0249] 3.2.2 G2 DBOP Br and 2K PEG Initiated pHPMA Core
[0250] In a typical reaction, G2 DBOP Br (0.324 g, 0.35 mmol) and
2K PEG initiator (0.745 g, 0.35 mmol) (for a targeted ratio of G2
dendron:750 PEG of 50:50 mol %) were weighed into a round bottom
flask, followed by HPMA (5.0 g, 34.7 mmol). EGDMA (112 .mu.l, 0.59
mmol) was added and the flask was equipped with magnetic stirrer
bar, sealed and degassed by bubbling with N.sub.2 for 20 minutes
and maintained under N.sub.2 at 30.degree. C. Anhydrous methanol
was degassed separately and subsequently added to the
monomer/initiator/brancher mixture via syringe to give a 50% v/v
mixture with respect to the monomer. The catalytic system; Cu(I)Cl
(0.069 g, 0.69 mmol) and 2,2'-bipyridyl (bpy) (0.217 g, 1.39 mmol),
were added under a positive nitrogen flow in order to initiate the
reaction. The polymerisations were stopped when conversions had
reached over 98%. The polymerisations were stopped by diluting with
a large excess of tetrahydrofuran (THF), which caused a colour
change from dark brown to a bright green colour. The catalytic
system was removed using Dowex.RTM. Marathon.TM. MSC (hydrogen
form) ion exchange resin beads and basic alumina. The resulting
polymer was isolated by precipitation from the minimum amount of
THF into cold hexane. The [initiator]:[CuCl]:[bpy] molar ratios in
all polymerizations were 1:1:2.
[0251] 3.2.3 G1 tBOC Dendron and Lactose Initiated pHPMA Core
[0252] The G1 .sup.tBOC Dendron initiator (48.5 mg, 0.09 mmol) and
Lactose ATRP initiator (70.7 mg, 0.09 mmol) was added to a 25 mL
round bottom flask equipped with a magnetic stirrer bar, followed
by the addition of 2,2-bipyridyl (56.2 mg, 0.360 mmol), EGDMA (28.5
mg, 0.144 mmol) and HPMA (1.3 g, 9.0 mmol). The reaction mixture
was bubbled with N.sub.2 for 15 minutes. Degassed anhydrous
methanol (3.3 mL) was added to the flask, and its contents stirred
and bubbled with N.sub.2 for a further 5 minutes. Copper (I)
chloride (17.8 mg, 0.180 mmol) was quickly weighed out and added to
the flask, instantly forming a brown coloured mixture, which was
stirred and bubbled with N.sub.2 for a further 5 minutes. A N.sub.2
pressure was built up within the flask, then N.sub.2 inlet then
removed, and the flask stirred for 24 hours at 40.degree. C. Once
the polymerisation was complete, THF was added to the reaction
flask to poison the Cu (I) catalyst, forming a green coloured
solution. The solution was passed through an alumina (neutral)
column to remove the catalytic system, concentrated in vacuo, and
precipitated into hexane. The supernatant was decanted off, and the
remaining white solid dried overnight in a vac-oven.
[0253] 3.2.4 G tBOC Dendron and Bifunctional Initiator pHPMA
Dumbbell Core
[0254] The G1 .sup.tBOC Dendron initiator (181 mg, 0.336 mmol) and
bi-functional initiator (36.6 mg, 0.084 mmol) was added to a 25 mL
round bottom flask equipped with a magnetic stirrer bar, followed
by the addition of 2,2-bipyridyl (157.4 mg, 1.01 mmol), EGDMA (79.1
mg, 0.399 mmol) and HPMA (3.63 g, 25.2 mmol). The reaction mixture
was then bubbled with N.sub.2 for 15 minutes. Degassed anhydrous
methanol (10 mL) was added to the flask, and its contents stirred
and bubbled with N.sub.2 for a further 5 minutes. Copper (I)
chloride (49.9 mg, 0.504 mmol) was quickly weighed out and added to
the flask, instantly forming a brown coloured mixture, which was
stirred and bubbled with N.sub.2 for a further 5 minutes. A N.sub.2
pressure was built up within the flask, then N.sub.2 inlet then
removed, and the flask stirred for 24 hours at 40.degree. C. Once
the polymerisation was complete, THF was added to the reaction
flask to poison the Cu (I) catalyst, forming a green coloured
solution. The solution was passed through an alumina (neutral)
column to remove the catalytic system, concentrated in vacuo, and
precipitated into hexane. The supernatant was decanted off, and the
remaining white solid dried overnight in a vac-oven.
[0255] 3.2.5 G2 tBOC Dendron and Bifunctional Initiator pHPMA
Dumbbell Core
[0256] The G2 .sup.tBOC Dendron initiator (197 mg, 0.168 mmol) and
bi-functional initiator (18.3 mg, 0.042 mmol) was added to a 25 mL
round bottom flask equipped with a magnetic stirrer bar, followed
by the addition of 2,2-bipyridyl (78.7 mg, 0.504 mmol), EGDMA (33.3
mg, 0.168 mmol) and HPMA (3.63 g, 12.6 mmol). The reaction mixture
was bubbled with N.sub.2 for 15 minutes. Degassed anhydrous
methanol (4.65 mL) was added to the flask, and its contents stirred
and bubbled with N.sub.2 for a further 5 minutes. Copper (I)
chloride (24.9 mg, 0.252 mmol) was quickly weighed out and added to
the flask, instantly forming a brown coloured mixture, which was
stirred and bubbled with N.sub.2 for a further 5 minutes. A N.sub.2
pressure was built up within the flask, then N.sub.2 inlet then
removed, and the flask stirred for 24 hours at 40.degree. C. Once
the polymerisation was complete, THF was added to the reaction
flask to poison the Cu (I) catalyst, forming a green coloured
solution. The solution was passed through an alumina (neutral)
column to remove the catalytic system, concentrated in vacuo, and
precipitated into hexane. The supernatant was decanted off, and the
remaining white solid dried overnight in a vac-oven.
TABLE-US-00002 TABLE 2 Mixed initiator polydendrons Polymer EGDMA
Initiator 1 Initiator 2 Core (mol %) Mn (gmol.sup.-1) Mw
(gmol.sup.-1) PDI G1 tBOC G2 tBOC pHPMA50 0.8 61500 153500 2.49 G1
tBOC Lactose pHPMA50 0.8 102000 216000 2.11 G1 tBOC bifunctional
pHPMA50 0.95 47000 227000 4.83 G2 tBOC bifunctional pHPMA50 0.8
177500 555500 3.13 G2 DBOP 750 PEG 100 0 pHPMA50 0.8 90 500 1 304
000 9.67 90 10 pHPMA50 0.8 68457 1495000 21.84 75 25 pHPMA50 0.8
52431 987762 18.88 50 50 pHPMA50 0.8 39447 480638 12.19 25 75
pHPMA50 0.8 36157 315320 8.73 10 90 pHPMA50 0.8 37672 286049 7.61 0
100 pHPMA50 0.8 68133 296179 4.35 25 75 pHPMA50 0.9 60738 675119
11.13 0 100 pHPMA50 0.95 74740 642728 8.60 G2 DBOP 2K PEG 100 0
pHPMA50 0.8 193576 2225000 11.49 90 10 pHPMA50 0.8 348067 2464000
7.08 75 25 PHPMA50 0.8 55050 1067000 19.38 50 50 pHPMA50 0.85 29372
709209 24.15 25 75 pHPMA50 0.95 141272 1862000 13.18 10 90 pHPMA50
0.95 40195 795274 19.79 0 100 pHPMA50 0.95 32246 476990 14.79 50 50
pHPMA100 0.8 79448 516794 6.51
4. Nanoprecipitation of Polydendrons
[0257] 4.1 Nanoparticle Formation (Slow Addition)--HR Method
[0258] In a typical procedure, 10 mg of sample was completely
dissolved in 2 mL of acetone at room temperature; the resulting
solution (5 mg mL.sup.-1) was added drop wise to 10 mL of distilled
water under vigorous stirring for ca. 15 min using a glass pipette.
The solution was stirred vigorously for 24 h at room temperature,
until the acetone was completely evaporated as determined by
.sup.1H NMR analysis, where no peak at .delta. 2.22 corresponding
to acetone was observed.
[0259] 4.2 Nanoprecipitation (Fast Addition)
[0260] Polydendrons were dissolved in THF for a minimum of 6 hours
at various concentrations. Once fully dissolved polymer in THF (1
ml, 5 mg/ml) was added quickly to a vial of water (5 ml) stirring
at 30.degree. C. The solvent was allowed to evaporate overnight in
a fume cupboard to give a final concentration of 1 mg/ml polymer in
water. By adjusting the starting concentration and the volume of
water used, the size of the corresponding nanoparticles can be
controlled to an extent. The nanoparticles formed were analysed by
dynamic light scattering (DLS) and fluorimetry.
TABLE-US-00003 TABLE 3 DLS data for 100% Dendron initiated
polydendrons EGDMA Initiator Polymer core (mol %) Size (d nm) PDI
G1 DBOP pHPMA20 0.8 61.72 0.117 G1 DBOP pHPMA50 0.8 63.9 0.130 G1
DBOP pHPMA100 0.8 69.89 0.070 G2 DBOP pHPMA20 0.8 81.33 0.076 G2
DBOP pHPMA50 0.8 80.78 0.083 G2 DBOP pHPMA100 0.8 80.56 0.119 G1-A
tamine pHPMA50 0.7 70.6 0.366 G1-A tamine ptBuMA50 0.95 45.98 0.217
G1-A tamine pDEAEMA50 0.9 136.2 0.148 G1-A tamine pOEGMA50 0.95
44.98 0.519 G0-D tamine pDEAEMA50 0.9 G1-D tamine pDEAEMA50 0.9
G2-D tamine pDEAEMA50 0.9 115.9 0.158 G2-D tamine pDEAEMA50-block-
0.9 162.9 0.082 tBuMA-st-EGDMA Xant G1 - post modified with; benzyl
pHPMA50 0.85 141.1 0.238 n-morpholino pHPMA50 0.85 159.3 0.166
PEG480 pHPMA50 0.85 106.9 0.257 PEG5000 pHPMA50 0.85 156.8
0.427
TABLE-US-00004 TABLE 4 DLS data for mixed initiator polydendrons
Polymer EGDMA Size Initiator 1 Initiator 2 core (mol %) (d nm) PDI
G1 tBOC bifunctional pHPMA50 0.95 73.78 0.109 G2 tBOC bifunctional
pHPMA50 0.8 27.33 0.116 G2 DBOP 750 PEG 100 0 pHPMA50 0.8 80.78
0.083 90 10 pHPMA50 0.8 115.6 0.069 75 25 pHPMA50 0.8 109.8 0.073
50 50 pHPMA50 0.8 114.6 0.067 25 75 pHPMA50 0.8 92.57 0.078 10 90
pHPMA50 0.8 94.26 0.091 0 100 pHPMA50 0.8 87.8 0.076 0 100 pHPMA50
0.95 89.53 0.083 G2 DBOP 2K PEG 100 0 pHPMA50 0.8 62.15 0.391 90 10
pHPMA50 0.8 144.4 0.036 75 25 pHPMA50 0.8 214.6 0.085 50 50 pHPMA50
0.85 105.5 0.058 25 75 pHPMA50 0.95 52.17 0.277 10 90 pHPMA50 0.95
37.81 0.207 0 100 pHPMA50 0.95 36.18 0.24 50 50 pHPMA20 0.85 54.9
0.296 50 50 pHPMA100 0.8 232.2 0.133
5. Encapsulation of Fluorescent Molecules
[0261] 5.1 Nile Red Encapsulation--HR Method
[0262] In a typical procedure, 10 mg of sample and 0.1 mg Nile Red
was dissolved completely in 2 mL of acetone at room temperature;
the resulting solution (5.05 mg mL.sup.-1) was added drop wise to
10 mL of distilled water under vigorous stirring for ca. 15 min
using a glass pipette. The solution was stirred vigorously for 24 h
at room temperature, until the acetone was completely evaporated as
determined by .sup.1H NMR analysis, where no peak at .delta. 2.22
corresponding to acetone was observed.
[0263] 5.2 Fluoresceinamine Encapsulation--HR Method
[0264] In a typical procedure, 10 mg of sample and 1 mg of
fluoresceinamine was dissolved completely in 2 mL of acetone at
room temperature; the resulting solution (5.5 mg mL.sup.-1) was
added drop wise to 10 mL of distilled water under vigorous stirring
for ca. 15 min using a glass pipette. The solution was stirred
vigorously for 24 h at room temperature, until the acetone was
completely evaporated as determined by .sup.1H NMR analysis, where
no peak at .delta. 2.22 corresponding to acetone was observed.
[0265] 5.3 Encapsulation of Nile Red or Pyrene Using Mixed
Initiator Polydendrons
[0266] Stock solutions of nile red in THF at 0.2 mg/ml and pyrene
in THF at 0.5 mg/ml were made. In a typical experiment the desired
amount of nile red or pyrene was added to a vial using a pipette
(e.g for a stock solution at 0.2 mg/ml, 100 .mu.l would be used if
0.02 mg was required). The vial was left in the fumecupboard for
.about.20 min to allow evaporation of the THF. A pre-dissolved
sample of polymer in THF (1 ml, 5 mg/ml) was added to the vial. The
vial was shaken gently to allow dissolution of the fluorescent
molecule in the THF containing polymer. Once the desired amount of
polymer and fluorescent molecule was dissolved in the I ml of THF,
this was added quickly to a vial of water (5 ml) stirring at
30.degree. C. The solvent was allowed to evaporate in a fume
cupboard overnight, giving a final concentration of I mg/ml polymer
in water. The nanoparticles formed were analysed by dynamic light
scattering (DLS) and fluorimetry.
[0267] Table 5 shows data for polymer nanoparticles with a final
concentration of 1 mg/ml polymer with 0.1 w/w % nile red or pyrene
encapsulated (1 .mu.g/ml)
TABLE-US-00005 TABLE 5 Fluorimetry of nanoparticles with nile red
and pyrene encapsulated Nile red encapsula- Pyrene tion (max
encapsu- Initiator 1 Initiator 2 Polymer EGDMA intensity at lation
G2 DBOP 750 PEG core (mol %) 630 mm) I1/I3 ratio 100 0 pHPMA50 0.8
702.1693 1.42 90 10 pHPMA50 0.8 625.9234 1.4458 75 25 pHPMA50 0.8
574.7425 1.4666 50 50 pHPMA50 0.8 548.357 1.4685 25 75 pHPMA50 0.8
243.2502 1.479 10 90 pHPMA50 0.8 404.1123 1.5208 0 100 pHPMA50 0.8
285.757 1.5315 0 100 pHPMA50 0.95 226.2446 --
6. Pharmacology
1. Materials & Methods
1. Materials
[0268] Dulbecco's Modified Eagles Medium (DMEM), Hanks buffered
saline solution (HBSS), Trypsin-EDTA, bovine serum albumin (BSA),
Nile red, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT reagent), acetonitrile (ACN) and all general
laboratory reagents were purchased from Sigma (Poole, UK). Foetal
bovine serum (FBS) was purchased from Gibco (Paisley, UK). The
CellTiter-Glo.RTM. Luminescent Cell Viability Assay kit was from
Promega (UK). The 24-well HTS transwell plates were obtained from
Corning (New York, USA). The 96-well black walled, flat bottomed
plates were from Sterilin (Newport, UK).
1.1 Routine Cell Culture/Cell Maintenance
[0269] Caco-2 cells were purchased from American Type Culture
Collection (ATCC, USA) and maintained in Dulbecco's Modified Eagles
Medium (DMEM) supplemented with 15% filtered sterile foetal bovine
serum. Cells were incubated at 37.degree. C. and 5% CO.sub.2 and
were routinely sub-cultured every 4 days when 90% confluent. Cell
count and viability was determined using a Countess automated cell
counter (Invitrogen).
1.2 Cytotoxicity
[0270] Caco-2 cells were seeded at a density of 1.0.times.10.sup.4
cells/100 .mu.l in DMEM supplemented with 15% FBS into each well of
a 96 well plate (Nunclon, Denmark) and incubated at 37.degree. C.
and 5% CO.sub.2. Cells from 4 separate flasks of biological
replicates of each cell type were used (N1-4) to improve
statistical power. Media was then aspirated from column I and
replaced with media containing each polydendron or aqueous Nile Red
solution at an equivalent 1 .mu.M Nile Red concentration then
diluted 1:1 in media across the plate up to column 11. Column 12
served as a negative control and consisted of media and untreated
cells. Following polydendron addition, the plates were incubated
for 24 hours or 120 hours at 37.degree. C., 5% CO.sub.2 prior to
assessment of cytotoxicity.
1.3 MTT Assay
[0271] Following incubation of treated plates for 24 h or 120 h, 20
.mu.l of 5 mg ml.sup.-1 MTT reagent was added to each well and
incubated for 2 hours. Subsequently, 100 .mu.L MTT lysis buffer
(50% N--N-Dimethylformamide in water containing 20% SDS, 2.5%
glacial acetic acid and 2.5% hydrochloric acid, pH 4.7) was added
to each well to lyse overnight at 37.degree. C., 5% CO.sub.2.
Following incubation the absorbance of each well was read using a
Tecan Genosis plate reader at 560 nm (Tecan Magellan, Austria).
1.4 ATP Assay
[0272] Following incubation of treated plates for 24 h or 120 h,
cells were equilibrated to room temperature for approximately 30
minutes. All but 20 .mu.l of media was removed from each well and
20 .mu.l CellTiter-Glo.RTM. (Promega, UK) reagent was added. All
reagents were made fresh and in accordance with the manufacturer's
instructions. Plates were put on an orbital shaker for 10 minutes
to mix contents and allow for stabilisation of luminescence signal.
Luminescence was then measured using a Tecan Genios plate reader
(Tecan Magellan, Austria).
2. Transcellular Permeability of Nile Red Across Caco-2
Monolayers
2.1 Setting Up and Treating Transwell Plates
[0273] Transwells were seeded with 3.5.times.10.sup.4 cells per
well and propagated to a monolayer over a 21 day period, during
which media in the apical and basolateral wells was changed every
other day. Trans-epithelial electrical resistance (TEER) values
were monitored until they were >1300.OMEGA.. 1 .mu.M of Nile Red
polydendron or 1 .mu.M aqueous Nile Red was added to the apical
chamber of 4 wells and the basolateral chamber of 4 wells to
quantify transport in both Apical to Basolateral (A>B) and
Basolateral to Apical (B>A) direction and sampled on an hourly
basis over a 4 h time period. Apparent permeability coefficient was
then determined by the amount of compound transported over time
using the equation:
Papp=(dQ/dt)(1/AC.sub.0)
where (dQ/dt) is the amount per time (nmolsec.sup.-1), A is the
surface area of the filter and C.sub.0 is the starting
concentration of the donor chamber (1 .mu.M).
2.3 Extraction and Quantification of Nile Red
[0274] 100 .mu.l of each collected sample was mixed with 900 .mu.l
acetone, vortexed, sonicated for 6 minutes and centrifuged at 13300
rpm for 3 minutes. The supernatant was completely dried in a vacuum
centrifuge at 30.degree. C. until the dry solid sample was left.
This was reconstituted in 150 .mu.l acetonitrile, transferred to a
96-well black walled, flat bottomed plate and measured for
fluorescence intensity excitation wavelength 480 nm, emission
wavelength 560 nm using a Tecan Genios plate reader (Tecan
Magellan, Austria).
3. Results
3.1 Cytotoxicity--MTT Assays
[0275] Following 24 hour incubation of Caco-2 cells with each
polydendron, analysis of cytotoxicity by MTT assay (FIG. 6) showed
that aqueous Nile Red and each polydendron did not affect metabolic
turnover of Caco-2 cells compared to untreated cells at the range
of concentrations investigated. It can be inferred that metabolic
turnover correlates to cell viability in which case each material
was not cytotoxic.
[0276] FIG. 6: MTT assay of Caco-2 cells following 24 hour
incubation with aqueous Nile Red and each polydendron. A=aqueous
Nile Red, EC.sub.50 1.160. B=0:100, EC.sub.50 2.509. C=10:90,
EC.sub.50 1.410. D=25:75, EC.sub.50 1.567. E=50:50, EC.sub.50
1.083. F=75:25, EC.sub.50 1.565, G=90:10, EC.sub.50 1.607. H=100:0,
EC.sub.50 2.678.
[0277] Following 120 hour incubation of Caco-2 cells with each
polydendron, analysis of cytotoxicity by MTT assay (FIG. 7) showed
that aqueous NR and each polydendron at the range of concentrations
investigated did not affect the viability of Caco-2 cells.
[0278] FIG. 7: MTT assay of Caco-2 cells following 120 hour
incubation with aqueous Nile Red and each polydendron. A=aqueous
Nile Red, EC.sub.50 No EC.sub.50. B=0:100, EC.sub.50 1.528.
C=10:90, EC.sub.50 No EC.sub.50. D=25:75, EC.sub.50 6.166. E=50:50,
EC.sub.50 0.7856. F=75:25, EC.sub.50 No EC.sub.50, G=90:10,
EC.sub.50 0.2176. H=100:0, EC.sub.50 No EC.sub.50.
3.3 ATP Assay
[0279] Following 24 hour incubation of Caco-2 cells with each
polydendron, analysis of cytotoxicity by ATP assay using a
CellTiter-Glo.RTM. kit (Promega, UK) (FIG. 8) indicated that ATP
presence was not affected in cells treated with aqueous Nile Red
solution and polydendron formulated Nile Red at the range of
concentrations investigated compared to untreated cells. It can be
inferred that the presence of ATP correlates to cell viability in
which case each material was not cytotoxic.
[0280] FIG. 8: ATP assay of Caco-2 cells following 24 hour
incubation with aqueous Nile Red and each polydendron. A=aqueous
Nile Red, EC.sub.50 1.946. B=0:100, EC.sub.50 2.855. C=10:90,
EC.sub.50 No EC.sub.50. D=25:75, EC.sub.50 No EC.sub.50. E=50:50,
EC.sub.50 No EC.sub.50. F=75:25, EC.sub.50 No EC.sub.50, G=90:10,
EC.sub.50 2.848. H=100:0, EC.sub.50 0.1961.
[0281] Following 120 hour incubation of Caco-2 cells with each
polydendron, analysis of cytotoxicity by ATP assay using a
CellTiter-Glo.RTM. kit (Promega, UK) (FIG. 9) indicated viability
was not affected in cells treated with aqueous Nile Red solution
and each polydendron material at the range of concentrations
investigated compared to untreated cells.
[0282] FIG. 9: ATP assay of Caco-2 cells following 120 hour
incubation with aqueous Nile Red and each polydendron. A=aqueous
Nile Red, EC.sub.50 No EC.sub.50. B=0:100, EC.sub.50 No EC.sub.50.
C=10:90, EC.sub.50 3.168. D=25:75, EC.sub.50 2.565. E=50:50,
EC.sub.50 No EC.sub.50. F=75:25, EC.sub.50 3.032, G=90:10,
EC.sub.50 No EC.sub.50. H=100:0, EC.sub.50 No EC.sub.50.
4. Transcellular Permeability of Selected Nile Red Polydendron
Materials Across Caco-2 Cell Monolayers.
[0283] Transcellular permeability of Nile Red through Caco-2 cell
monolayers (to model the intestinal epithelium) was significantly
higher in the apical to basolateral (A>B, gut to blood)
direction for the polydendron preparation 10G2:90PEG compared to an
aqueous solution of Nile Red (FIGS. 10 A&B). All the
polydendron materials produced a greater apical to basolateral
(A>B, gut to blood), basolateral to apical (B>A, blood to
gut) ratio than an aqueous preparation of Nile Red following 1 hour
incubation (Table 1, FIG. 10 C). A statistically significant
correlation (P=<0.05) between the ratio of dendron and PEG used
in the polydendron formulation and the ratio of apical to
basolateral (A>B, gut to blood), basolateral to apical (B>A,
blood to gut) movement of Nile Red across the Caco-2 monolayer was
observed (FIG. 10 C).
[0284] FIG. 10. (A&B) Transcellular permeability across Caco2
cell monolayers of polydendron formulated Nile Red relative to an
aqueous solution of Nile Red. Data are given as the mean of
experiments conducted in biological triplicate. (C) Correlation
between polydendron formulation and the ratio of Nile Red
transported (A>B/B>A) across Caco2 cell monolayers (r.sup.2
0.784). Data were normally distributed, statistical analysis was
conducted using a Pearson correlation (P=<0.05) a two-tailed P
value was used to reduce the chance of a type I error.
TABLE-US-00006 TABLE 1 Apparent permeability (Papp) of Nile Red
polydendrons and aqueous Nile Red across Caco2 cell monolayers
following 1 hour incubation. Data are given as the mean of
experiments conducted in biological triplicate. Papp (cm s.sup.-1)
Polydendron Formulation Apical > Basolateral > A > B/B
> A (G2:PEG ratio) Basolateral Apical ratio 1.00 1.763 .times.
10.sup.-5 1.538 .times. 10.sup.-6 11.4605 0.75 2.613 .times.
10.sup.-5 2.056 .times. 10.sup.-6 12.7123 0.50 5.271 .times.
10.sup.-5 5.555 .times. 10.sup.-6 9.4872 0.25 4.135 .times.
10.sup.-5 4.684 .times. 10.sup.-6 8.8279 0.10 4.042 .times.
10.sup.-4 4.580 .times. 10.sup.-5 8.8255 0.00 2.060 .times.
10.sup.-5 3.188 .times. 10.sup.-6 6.4626 Aqueous Nile Red 2.371
.times. 10.sup.-5 6.384 .times. 10.sup.-6 3.7140
7. Example of Nanoprecipitation to Encapsulate Inorganic Magnetic
Nanoparticles
[0285] Polydendron (G2:2K PEG(50:50)-pHPMA.sub.50-EGDMA.sub.0.8)
was dissolved in THF for a minimum of 6 hours. Once fully dissolved
the polymer in THF (0.2 ml, 25 mg/ml) was mixed with
Fe.sub.3O.sub.410 nm particles in THF (0.5 ml, 5 mg/ml) and this
mixture of polymer and Fe.sub.3O.sub.4 was added quickly to a vial
of water (1 ml) stirring at 30.degree. C. The solvent was allowed
to evaporate overnight in a fume cupboard to give a final
concentration of 5 mg/ml polymer, 2.5 mg/ml Fe.sub.3O.sub.4 in
water. The nanoparticles formed were analysed by dynamic light
scattering (DLS), scanning electron microscopy (SEM) and
transmission electron microscopy (TEM).
[0286] SEM imaging showed spherical nanoparticles of size range
varying from approximately 150 to 250 nm while TEM imaging showed
the majority of nanoparticles to have encapsulated Fe.sub.3O.sub.4
with no free Fe.sub.3O.sub.4 observed.
[0287] DLS (2.5 mg/ml in water) determined the Z-Ave hydrodynamic
diameter to be 182 nm with PDI to be 0.01. In the presence of a
magnetic field (i.e. with a magnetic suspended above, just touching
the surface of the dispersion) DLS measurements showed a 50%
reduction in derived count rate after 12 hours and a 40% reduction
in derived count rate after 8 hours, with Z-Ave diameter remaining
constant throughout. The reduction in derived count rate is
intrinsic to a decrease in concentration of nanoparticles within
the dispersion and demonstrates the effect of the magnetic field on
directing the behaviour of the nanoprecipitate. In the absence of a
magnetic field there is no drop in derived count rate.
* * * * *